Compositions and methods for modulating metabolic pathways

ABSTRACT

Compositions and methods useful for inducing an increase in fatty acid oxidation or mitochondrial biogenesis, reducing weight gain, inducing weight loss, or increasing Sirt1, Sirt3, or AMPK activity are provided herein. Such compositions can contain synergizing amounts of a sirtuin-pathway activators, including but not limited to resveratrol, in combination with beta-hydroxymethylbutyrate (HMB), keto isocaproic acid (KIC), leucine, or combinations of HMB, KIC and leucine.

CROSS-REFERENCE

This application is a continuation application which claims the benefitof U.S. application Ser. No. 14/746,516, filed Jun. 22, 2015; whichclaims the benefit of Ser. No. 13/866,936, filed Apr. 19, 2013, now U.S.Pat. No. 9,072,692, which claims the benefit of Ser. No. 13/549,381,filed Jul. 13, 2012, now U.S. Pat. No. 8,617,886, which claims thebenefit of U.S. Application No. 61/508,139, filed Jul. 15, 2011; U.S.Application No. 61/636,597, filed Apr. 20, 2012; U.S. Application No.61/636,598, filed Apr. 20, 2012; U.S. Application No. 61/636,603, filedApr. 20, 2012; U.S. Application No. 61/636,605, filed Apr. 20, 2012;U.S. Application No. 61/636,608, filed Apr. 20, 2012; U.S. ApplicationNo. 61/636,610, filed Apr. 20, 2012; all of which applications areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

All organisms have developed exquisite metabolic pathways that maintainenergy homeostasis by balancing their intake and metabolism of energywith their expenditure needs of the organism. In mammals, these pathwaysregulate food intake, glucose homeostasis, storage of energy in fatand/or muscle, and mobilization of energy by, for instance, physicalactivity. Malfunctioning of these pathways, often resulting from excessenergy intake relative to energy expenditure, leads to imbalance inenergy homeostasis and can lead to a wide range of metabolic disorders,such as obesity, diabetes, hypertension, arteriosclerosis, highcholesterol, and hyperlipidemia.

The high incidence of metabolic disorders in humans and their relatedimpact on health and mortality represents a significant threat to publichealth. For instance, obesity, clinically defined as a body mass indexof over 30 kg/m², is estimated to affect 35.7% of the U.S. adultpopulation. Obesity increases the likelihood of many diseases, such asheart disease and type II diabetes, which is one of the leadingpreventable causes of death worldwide. In the U.S., obesity is estimatedto cause roughly 110,000-365,000 deaths per year. Diabetes is ametabolic disorder characterized by high blood glucose levels or lowglucose tolerance, and is estimated to affect 8% of the U.S. population.Diabetes is also significantly associated with higher risk of death fromvascular disease, cancer, renal disease, infectious diseases, externalcauses, intentional self-harm, nervous system disorders, and chronicpulmonary disease (N Engl J Med 2011; 364:829-841). Metabolic syndrome,in which subjects present with central obesity and at least two othermetabolic disorders (such as high cholesterol, high blood pressure, ordiabetes), is estimated to affect 25% of the U.S. population.

Sirtuins are highly conserved protein deacetylases and/orADP-ribosyltransferases that have been shown to extend lifespan in lowermodel organisms, such as yeast, C. elegans, and drosophila. In mammals,sirtuins have been shown to act as metabolic sensors, responding toenvironmental signals to coordinate the activity of genes that regulatemultiple energy homeostasis pathways. For example, studies have shownthat sirtuin activation mimics the effects of caloric restriction, anintervention demonstrated to significantly extend lifespan, andactivates genes that improve glucose homeostasis and the conversion offat to energy by fatty acid oxidation.

Many efforts have been attempted to develop treatments for metabolicdisorders by targeting specific energy metabolism pathways. Theseefforts have resulted in the development of, for example, isoflavones(U.S. Patent Application No. 20110165125), tetrahydrolipstatin (U.S.Pat. No. 6,004,996), and compositions that modulate the SIRT1 and AMPKpathways (U.S. Patent Application Nos. 20100210692, 20100009992,20070244202 and 20080176822). However, these efforts are of limitedsuccess. For instance, use of the SIRT1 activator resveratrol in humansis hampered by its limited bioavailability, necessitating high dosageswhich have raised safety concerns. Thus, there remains a great need fortreatments that can address a wide range of metabolic disorders bysafely regulating metabolic pathways.

SUMMARY OF THE INVENTION

The subject application provides compositions useful for inducing anincrease in fatty acid oxidation and mitochondrial biogenesis in asubject. The compositions also cause activation of Sirt1 and Sirt3,thereby mediating beneficial downstream effects, including preventionand treatment of diabetes, cardiovascular disease and inflammatorydisease. Such compositions contain synergizing amounts of a sirtuinpathway activator (e.g. resveratrol) in combination with a branchedchain amino acid and/or metabolites thereof (e.g.beta-hydroxymethylbutyrate (HMB), leucine, keto-isocaproic acid (KIC) orcombinations of HMB, KIC and/or leucine). The subject application alsoprovides methods of increasing fatty acid oxidation in a subjectcomprising the administration of the disclosed compositions.

The invention provides for a composition comprising: (a) one or moretypes of branched amino acids (e.g. leucine) and/or metabolites thereofand (b) a sirtuin-pathway activator that optionally may be present in asub-therapeutic amount, wherein the composition is synergisticallyeffective in increasing the sirtuin-pathway output by at least about1-fold (e.g. at least about 1, 2, 3, 4, 5 or more fold) as compared tothat of component (a) or (b) when it is being used alone. In someembodiments, the synergistic effect is observed when (i) media frommyotybes or adipocytes treated with the composition is administered tothe other of the myotubes or adipocytes, (ii) the composition isadministered to myotubes or adipocytes, and/or (iii) the composition isadministered to a subject.

In some embodiments of any aspect described herein, an increase insirtuin-pathway output is evidenced by an increase in physiologicaleffect selected from the group consisting of mitochondrial biogenesis,fatty acid oxidation, glucose uptake, palmitate uptake, oxygenconsumption, carbon dioxide production, weight loss, heat production,visceral adipose tissue loss, respiratory exchange ratio, insulinsensitivity, a inflammation marker level, body temperature, fat cellbrowning, irisin production, and vasodilatation. An increase insirtuin-pathway output can be evidenced by an increase in expression oractivity level of one or more of the group consisting of SIRT1, SIRT3,and PGC1-α. The increase in sirtuin-pathway output can be at least about1, 3, 5, 6, 8, 10, 15, 20, or 50 fold.

Another aspect of the invention provides for a composition comprising:(a) one or more types of branched amino acids (e.g. leucine) and/ormetabolites thereof, and (b) a sirtuin-pathway activator, wherein molarratio of component (a) to (b) in said composition is greater than about20, and wherein the composition when administered to a subject in needthereof synergistically enhances mitochondrial biogenesis as measured bya decrease in weight gain of a subject, a decrease in visceral adiposevolume of a subject, an increase in fat oxidation of a subject, anincrease in irisin production of a subject, an increase in insulinsensitivity of a subject, an increase of glucose uptake in muscle of asubject, a decrease in inflammation markers, an increase invasodilatation, and/or an increase in body temperature. In someembodiments, the molar ratio of component (a) to (b) in said compositionis greater than about 5, 10, 15, 20, 25, 30, 35, 40, 60, 80, 100, 150,200, 250, or more.

Another aspect of the invention provides for a composition comprising: aunit dosage suitable for oral ingestion, said unit dosage comprising:(a) one or more types of branched amino acids (e.g. leucine) and/ormetabolites thereof, and (b) a substantially homogeneous population ofpolyphenol or polyphenol precursor molecules, and wherein the unitdosage is effective in inducing an increase in sirtuin pathway output asmeasured by a decrease in weight gain of a subject, a decrease invisceral adipose volume of a subject, an increase in fat oxidation of asubject, an increase in insulin sensitivity of a subject, an increase ofglucose uptake in muscle of a subject, an increase in vasodilatation,and/or an increase in body temperature. In some embodiments, the unitdosage is formulated as a tablet, capsule, or gel capsule.

The polyphenol or polyphenol precursor molecules can be present in anamount effective in increasing sirtuin-pathway output (e.g. by about,less than about, or more than about 1-fold, 3-fold, 5-fold, 6-fold,8-fold, 10-fold, 15-fold, 20-fold, 50-fold, or more). The polyphenol orpolyphenol precursor molecules can be present in an amount effective insirtuin-pathway output by at least about 1, 2, 3, 4, 5 or more fold. Thepolyphenol molecules can activate SIRT1 and/or SIRT3. The polyphenol canactivate AMPK. The polyphenol can activate PGC1α. The polyphenol can beresveratrol or an analog thereof. The polyphenol can be chlorogenicacid. The polyphenol can be selected from the group consisting ofchlorogenic acid, resveratrol, caffeic acid, quinic acid, piceatannol,ellagic acid, epigallocatechin gallate, grape seed extract, cinnamicacid, ferulic acid, and any analog thereof.

Another aspect of the invention provides for a food compositioncomprising: (a) one or more types of branched amino acids (e.g. leucine)and/or metabolites thereof; (b) a sirtuin pathway activator, wherein (a)and (b) are present in an amount that synergistically effect an increasein sirtuin pathway output as measured by a decrease in weight gain of asubject, a decrease in visceral adipose volume of a subject, an increasein fat oxidation of a subject, an increase in insulin sensitivity of asubject, an increase of glucose uptake in muscle of a subject, anincrease in vasodilatation, and/or an increase in body temperature; and(c) a food carrier.

The composition can be a dietary supplement packaged as a liquid (e.g. abeverage), solid (e.g. solid food) or semi-solid (e.g. semi-solid food).In some embodiments, the food carrier is a juice, coffee, tea, soda, orsnack bar. The composition can be formulated as an oral dosage form. Thecomposition can be packaged as a unit dosage. The unit dosage can beformulated as a tablet, capsule, or gel capsule.

Another aspect of the invention provides for a composition comprising: asynergistically effective amount of (a) one or more types of branchedamino acids (e.g. leucine) and/or metabolites thereof; and (b) asirtuin-pathway activator, wherein the composition is substantially freeof non-branched amino acids, wherein the combination when administeredto a subject in need thereof enhances mitochondrial biogenesis to agreater degree as compared to administering to a subject component (a)or component (b) alone, and wherein the enhanced mitochondrialbiogenesis is measured by a decrease in weight of a subject, a decreasein visceral adipose volume of a subject, an increase in fat oxidation ofa subject, an increase in insulin sensitivity of a subject, an increaseof glucose uptake in muscle of a subject, an increase in vasodilatation,and/or an increase in body temperature. The increase mitochondrialbiogenesis can be at least about 1, 2, 3, 4, 5, 6, 8, 10, 15, 20, or 50fold (where a 1-fold increase is equivalent to a 100% increase). In someembodiments, the change in mitochondrial biogenesis and/or in one ormore measures thereof is about or more than about 10%, 20%, 50%, 100%,200%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000%, or more.

Another aspect of the invention provides for a composition comprising:(a) one or more types of branched amino acids (e.g. leucine) and/ormetabolites thereof, and (b) a signaling molecule downstream of PGC1α ina sirtuin-signaling pathway. The signaling molecule downstream of PGC1αcan be irisin or an analog thereof. In some embodiments, the one or moretypes of branched amino acids and/or metabolites thereof can be selectedfrom the group consisting of leucine, valine, isoleucine,4-hydroxyisoleucine, keto-isocaproic acid (KIC),alpha-hydroxy-isocaproic acid, and HMB. The composition is substantiallyfree of non-branched amino acids.

In one aspect, the invention provides a composition comprising: (a) oneor more types of branched amino acids (e.g. leucine) and/or metabolitesthereof, and (b) a sub-therapeutic amount of one or more anti-diabeticagents selected from the group consisting of biguanide, meglitinide,sulfonylurea, thiazolidinedione, alpha glucosidase inhibitor, and ergotalkaloid; wherein the combination when administered to a subjectsynergistically increases insulin sensitivity in said subject ascompared to administering to a subject component (a) or component (b)alone. In some embodiments, the anti-diabetic agent is a sirtuin pathwayactivator. In some embodiments, the anti-diabetic agent is a biguanide(e.g. metformin or any analog thereof). In some embodiments, theincrease in insulin sensitivity is at least about a 1-fold increase(e.g. at least about 1, 2, 3, 4, 5, 6, 8, 10, 15, 20, or 50 fold). Insome embodiments, the invention provides a method of potentiating thetherapeutic efficacy of a biguanide comprising administeringsimultaneously or sequentially to a subject component (a) and component(b) of a composition of the invention, wherein the administration of (a)and (b) is in an amount that synergistically increases insulinsensitivity, and wherein component (b) is a biguanide (e.g. metformin).

The invention also provides for a method of potentiating the therapeuticefficacy of one or more anti-diabetic agents selected from the groupconsisting of biguanide, meglitinide, sulfonylurea, thiazolidinedione,alpha glucosidase inhibitor, and ergot alkaloid, comprisingadministering simultaneously or sequentially to a subject (a) asub-therapeutic amount of said anti-diabetic agent, and (b) one or morebranched amino acids, wherein the administration of (a) and (b) iseffective in ameliorating a diabetic symptom of said subject. Examplesof diabetic symptoms include, but are not limited to, polyuria,polydipsia, weight loss, polyphagia, blurred vision, hypertension,abnormalities of lipoprotein metabolism, and periodontal disease. Thebiguanide can be metformin. The one or more anti-diabetic agent cancomprise glipizide and/or metformin. The one or more anti-diabetic agentcan be thiazolidinedione.

In one aspect, the invention provides a method of increasing a level ofirisin, such as increasing production of irisin by a cell or in asubject. In some embodiments, the method comprises administering acomposition comprising: (a) one or more types of branched amino acids(e.g. leucine) and/or metabolites thereof, and (b) a sirtuin pathwayactivator; wherein the administering increases production of irisin by acell. In some embodiments, the increase in irisin production (or in anindicator providing evidence thereof) is an increase of about, less thanabout, or more than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 125%, 150%, 175%, 200%, or more. In some embodiments, theincrease in irisin production (or in an indicator providing evidencethereof) is an increase of about, less than about, or more than about1-fold, 3-fold, 5-fold, 6-fold, 8-fold, 10-fold, 15-fold, 20-fold,50-fold, or more. In some embodiments, the increase in irisin productionis evidenced by an increase in FNDC5 expression (e.g. as measured frommRNA and/or protein level). In some embodiments, the increase in irisinproduction is evidenced by an increase in one or more indicia of fatcell browning (e.g. fatty acid oxidation, and/or an increase inexpression of one or more brown fat selective genes in adipose tissue).In some embodiments, the increase in irisin production is evidenced byincreased secretion of irisin from the cell or in the subject (e.g. asmeasured from media in which the cell is cultured, or from circulatingplasma in a subject). In some embodiments, the composition comprisesleucine and resveratrol. In some embodiments, the composition comprisesleucine and cinnamic acid. In some embodiments, the compositioncomprises HMB and resveratrol. In some embodiments, the compositioncomprises HMB and cinnamic acid.

In some embodiments of any of the aspects described herein, thecomposition is suitable for oral consumption. The composition can be aliquid form suitable for non-oral administration to a subject. Thecomposition can be a liquid form suitable for injectable administrationto a subject. The composition can be formulated for oral administrationto a subject.

The invention provides for a method of enhancing fat oxidation in asubject in need thereof comprising administering to the subject any ofthe compositions described herein over a time period, wherein the fatoxidation in the subject is increased over the time period. Theinvention provides for a method of reducing an inflammatory response ina subject in need thereof comprising administering to the subject any ofthe compositions described herein over a time period, wherein theinflammatory response in the subject is reduced over the time period.The invention provides for a method of increasing or maintaining bodytemperature in a subject comprising administering to the subject any ofthe compositions described herein over a time period, wherein the bodytemperature in the subject is increased over the time period. Theinvention provides for a method of inducing vasodilatation comprisingadministering to the subject any of the compositions described hereinover a time period, wherein the vasodilation in the subject is inducedover the time period. The invention provides for a method of treatingdiabetes, comprising administering to the subject any of thecompositions described herein over a time period, wherein the insulinsensitivity in the subject is increased over the time period. In someembodiments, an increase in insulin sensitivity is evidenced by adecrease in plasma insulin levels, and/or an increase in glucoseutilization (e.g. faster glucose uptake in response to glucosechallenge). In some embodiments, the increase in fat oxidation and/orthe increase in insulin sensitivity is about, less than about, or morethan about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%,150%, 175%, 200%, or more. In some embodiments, the increase in fatoxidation and insulin sensitivity is more than about 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, or more. Insome embodiments, the increase in fat oxidation and/or the increase ininsulin sensitivity is about, less than about, or more than about1-fold, 3-fold, 5-fold, 6-fold, 8-fold, 10-fold, 15-fold, 20-fold,50-fold, or more. In some embodiments, the increase in fat oxidationand/or the increase in insulin sensitivity is more than about 1-fold,3-fold, 5-fold, 6-fold, 8-fold, 10-fold, 15-fold, 20-fold, 50-fold, ormore.

The invention provides for a method of preparing a composition of any ofthe compositions described herein, comprising mixing the components toform a substantially homogeneous mixture and forming the compositioninto a unit dosage.

In some embodiments of any of the aspects described herein, the one ormore types of branched amino acids and/or metabolites thereof isselected from the group consisting of leucine, valine, isoleucine,4-hydroxyisoleucine, keto-isocaproic acid (KIC),alpha-hydroxy-isocaproic acid, and hydroxymethylbutyrate (HMB). Thecomposition can be substantially free of non-branched amino acids. Thecomposition can comprise at least about 500 mg leucine and/or at leastabout 200 mg of the one or more metabolites.

In some embodiments of any of the aspects described herein, thesirtuin-pathway activator can activate one or more of SIRT1, SIRT3,AMPK, and PGC1α. In some embodiments, the sirtuin-pathway activator is apolyphenol or polyphenol precursor. In some embodiments, thesirtuin-pathway activator is resveratrol or an analog thereof. Thepolyphenol can be chlorogenic acid. The polyphenol or polyphenolprecursor can be selected from the group consisting of chlorogenic acid,resveratrol, caffeic acid, cinnamic acid, ferulic acid, piceatannol,ellagic acid, epigallocatechin gallate, grape seed extract, and anyanalog thereof. The sirtuin-pathway activator can be selected from thegroup consisting of cinnamic acid, quinic acid, fucoxanthin, abiguanide, rosiglitazone, or any analog thereof. The biguanide can bemetformin.

In some embodiments of any of the aspects described herein, thecomposition has one or more additional characteristics. In someembodiments, the composition is a food composition. The composition canbe a food or a dietary supplement packaged as a liquid (e.g. abeverage), a solid (e.g. solid food), or a semi-solid (e.g. semi-solidfood). In some embodiments, the composition is formulated as an oraldosage form. In some embodiments, the composition can be packaged as aunit dosage. The unit dosage can be formulated as a tablet, capsule, orgel capsule. In some embodiments, the composition further comprises apharmaceutically active agent. In some embodiments, the compositionfurther comprises an anti-diabetic agent. The composition can be apharmaceutical composition further comprising a pharmaceuticallyacceptable excipient. In some embodiments, administration of acomposition to a subject synergistically increases mitochondrialbiogenesis by at least about 1-fold, 3-fold, 5-fold, 6-fold, 8-fold,10-fold, 15-fold, 20-fold, or 50-fold, or more. In some embodiments,administration of a composition to a subject synergistically increasessirtuin pathway output by at least about 1-fold, 3-fold, 5-fold, 6-fold,8-fold, 10-fold, 15-fold, 20-fold, or 50-fold, or more.

Furthermore, the following non-limiting embodiments are also provided:

The invention provides for a composition comprising a synergizing amountof resveratrol, a synergizing amount of beta-hydroxymethylbutyrate(HMB), a synergizing amount of keto isocaproic acid (KIC); and/or asynergizing amount of leucine. In some embodiments, the compositioncomprises a synergizing amount of resveratrol and a synergizing amountof HMB, wherein: said synergizing amount of resveratrol is between atleast 35 mg and about 500 mg, and said synergizing amount of HMB isbetween about (or at least) 0.20 g and about 3.0 g.

In some embodiments, the composition comprises a synergizing amount ofresveratrol and a synergizing amount of leucine, wherein: saidsynergizing amount of resveratrol is between at least 35 mg and about500 mg; and said synergizing amount of leucine is between about (or atleast) 0.75 g and about 3.0 g. The composition can comprise asynergizing amount of resveratrol and a synergizing amount of KIC,wherein: said synergizing amount of resveratrol is between at least 35mg and about 500 mg; and said synergizing amount of KIC is between about(or at least) 0.75 g and about 3.0 g. The composition can comprise asynergizing amount of resveratrol, a synergizing amount of HMB and asynergizing amount of leucine, provided that the total amount of HMB andleucine in said composition is less than (or less than about) 3.0 g,wherein: said synergizing amount of resveratrol is between at least 35mg and about 500 mg; said synergizing amount of HMB is between about (orat least) 0.20 g and about 3.0 g; and said synergizing amount of leucineis between about (or at least) 0.50 g and about 3.0 g. The compositioncan comprise a synergizing amount of resveratrol, a synergizing amountof KIC and a synergizing amount of leucine, provided that the totalamount of KIC and leucine in said composition is less than (or less thanabout) 3.0 g, wherein: said synergizing amount of resveratrol is betweenat least 35 mg and about 500 mg; said synergizing amount of KIC isbetween about (or at least) 0.50 g and about 3.0 g; and said synergizingamount of leucine is between about (or at least) 0.50 g and about 3.0 g.The composition can comprise a synergizing amount of resveratrol, asynergizing amount of HMB and a synergizing amount of KIC, provided thatthe total amount of HMB and KIC in said composition is less than (orless than about) 3.0 g, wherein: said synergizing amount of resveratrolis between at least 35 mg and about 500 mg; said synergizing amount ofHMB is between about (or at least) 0.20 g and about 3.0 g; and saidsynergizing amount of KIC is between about (or at least) 0.50 g andabout 3.0 g.

The composition can comprise a synergizing amount of resveratrol, asynergizing amount of KIC, a synergizing amount of HMB and a synergizingamount of leucine, provided that the total amount of KIC, HMB andleucine in said composition is less than (or less than about) 3.0 g,wherein: said synergizing amount of resveratrol is between at least 35mg and about 500 mg; said synergizing amount of HMB is between about (orat least) 0.20 g and about 3.0 g; said synergizing amount of KIC isbetween about (or at least) 0.50 g and about 3.0 g; and said synergizingamount of leucine is between about (or at least) 0.50 g and about 3.0 g.

In some embodiments, said synergizing amount of resveratrol is betweenat least 50 mg and about 500 mg; and said synergizing amount of HMB isbetween about (or at least) 0.40 g and about 3.0 g. In otherembodiments, said synergizing amount of resveratrol is between at least50 mg and about 500 mg; and said synergizing amount of leucine isbetween about (or at least) 0.75 g and about 3.0 g. In some embodiments,said synergizing amount of resveratrol is between at least 50 mg andabout 500 mg; said synergizing amount of HMB is between at least 0.40 gand about 3.0 g; and said synergizing amount of leucine is between atleast 0.75 g and about 3.0 g. In other embodiments, said synergizingamount of resveratrol is between at least 50 mg and about 500 mg; saidsynergizing amount of KIC is between at least 0.75 g and about 3.0 g;and said synergizing amount of leucine is between at least 0.75 g andabout 3.0 g. In some embodiments, said synergizing amount of resveratrolis between at least 50 mg and about 500 mg; said synergizing amount ofHMB is between at least 0.40 g and about 3.0 g; and said synergizingamount of KIC is between at least 0.75 g and about 3.0 g.

In some embodiments, said synergizing amount of resveratrol is betweenat least 50 mg and about 500 mg; said synergizing amount of HMB isbetween at least 0.40 g and about 3.0 g; said synergizing amount of KICis between at least 0.75 g and about 3.0 g; and said synergizing amountof leucine is between at least 0.75 g and about 3.0 g.

In some of the embodiments described herein, the amount or HMB, KIC,leucine or combinations of leucine, KIC and/or HMB may be less than, orequal to, 3.0 g.

In some of the embodiments described herein, said composition mayexclude one or more of the amino acids selected from the groupconsisting of lysine, glutamate, proline, arginine, valine, isoleucine,aspartic acid, asparagine, glycine, threonine, serine, phenylalanine,tyrosine, histidine, alanine, tryptophan, methionine, glutamine,taurine, carnitine, cystine and cysteine.

In some of the embodiments described herein, the composition may excludeone or more of the following ingredients: niacin, vitamin B6, vitaminB12, pantothenic acid, caffeine, green tea extract, extracts fromguarana seed or extracts from guarana plants.

In some of the embodiments described herein, said composition mayexclude one or more of the amino acids selected from the groupconsisting of lysine, glutamate, proline, arginine, valine, isoleucine,aspartic acid, asparagine, glycine, threonine, serine, phenylalanine,tyrosine, histidine, alanine, tryptophan, methionine, glutamine,taurine, carnitine, cystine and cysteine.

In some of the embodiments described herein, the composition may excludeone or more of the following ingredients: niacin, vitamin B6, vitaminB12, pantothenic acid, caffeine, green tea extract, extracts fromguarana seed or extracts from guarana plants.

In some of the embodiments described herein, said composition mayexclude one or more of the amino acids selected from the groupconsisting of lysine, glutamate, proline, arginine, valine, isoleucine,aspartic acid, asparagine, glycine, threonine, serine, phenylalanine,tyrosine, histidine, alanine, tryptophan, methionine, glutamine,taurine, carnitine, cystine and cysteine. In some embodiments, thecomposition excludes valine and/or isoleucine.

In some of the embodiments described herein, the composition may furthercomprise a flavorant. In any one of the embodiments described herein,said composition is a solid, liquid, emulsion, gel or paste.

The invention provides for a method of increasing fatty acid oxidationin a subject comprising the administration of a composition comprising asynergizing amount of resveratrol, a synergizing amount ofbeta-hydroxymethylbutyrate (HMB), a synergizing amount of ketoisocaproic acid (KIC), and/or a synergizing amount of leucine to asubject in an amount effective to increase fatty acid oxidation.

In one aspect, the invention provides for a method of reducing weightgain or inducing weight loss in a subject comprising the administrationof a composition comprising a synergizing amount of resveratrol, asynergizing amount of beta-hydroxymethylbutyrate (HMB), a synergizingamount of keto isocaproic acid (KIC), and/or a synergizing amount ofleucine to a subject in an amount effective to reduce weight gain orinduce weight loss.

In another aspect, the invention provides for a method of stimulatingSirt1 or Sirt3 comprising the administration of a composition comprisinga synergizing amount of resveratrol, a synergizing amount ofbeta-hydroxymethylbutyrate (HMB), a synergizing amount of ketoisocaproic acid (KIC), and/or a synergizing amount of leucine to asubject in an amount effective to stimulate SIRT1 or SIRT3.

The invention provides for a method of activating the metabolic activityof adipocytes, smooth muscle, skeletal muscle or cardiac musclecomprising the administration of a composition comprising a synergizingamount of resveratrol, a synergizing amount ofbeta-hydroxymethylbutyrate (HMB), a synergizing amount of ketoisocaproic acid (KIC), and/or a synergizing amount of leucine to asubject in an amount sufficient to activate the metabolic activity ofsaid muscle.

In other embodiments, the invention provides for a method of increasingor maintaining body temperature in a subject comprising theadministration of a composition comprising a synergizing amount ofresveratrol, a synergizing amount of beta-hydroxymethylbutyrate (HMB), asynergizing amount of keto isocaproic acid (KIC), and/or a synergizingamount of leucine to a subject in an amount sufficient to increase ormaintain the body temperature of said subject.

The invention provides for a method of treating type 2 diabetes in asubject comprising the administration of a composition comprising asynergizing amount of resveratrol, a synergizing amount ofbeta-hydroxymethylbutyrate (HMB), a synergizing amount of ketoisocaproic acid (KIC), and/or a synergizing amount of leucine to asubject in an amount sufficient to treat type 2 diabetes in saidsubject.

The invention also provides for a method of reducing an inflammatoryresponse in a subject comprising the administration of a compositioncomprising a synergizing amount of resveratrol, a synergizing amount ofbeta-hydroxymethylbutyrate (HMB), a synergizing amount of ketoisocaproic acid (KIC), and/or a synergizing amount of leucine to asubject in an amount sufficient to reduce an inflammatory response insaid subject.

The invention provides for a method of inducing vasodilation comprisingthe administration of a composition comprising a synergizing amount ofresveratrol, a synergizing amount of beta-hydroxymethylbutyrate (HMB), asynergizing amount of keto isocaproic acid (KIC), and/or a synergizingamount of leucine in an amount sufficient to induce vasodilation in saidsubject.

In some embodiments, the composition comprises a synergizing amount ofresveratrol and a synergizing amount of HMB, wherein: said synergizingamount of resveratrol is between at least 35 mg and about 500 mg, andsaid synergizing amount of HMB is between about (or at least) 0.20 g andabout 3.0 g.

In some embodiments, the composition comprises a synergizing amount ofresveratrol and a synergizing amount of leucine, wherein: saidsynergizing amount of resveratrol is between at least 35 mg and about500 mg, and said synergizing amount of leucine is between about (or atleast) 0.75 g and about 3.0 g.

In other embodiments, the composition comprises a synergizing amount ofresveratrol and a synergizing amount of KIC, wherein: said synergizingamount of resveratrol is between at least 35 mg and about 500 mg, andsaid synergizing amount of KIC is between about (or at least) 0.75 g andabout 3.0 g.

In some embodiments, the composition comprises a synergizing amount ofresveratrol, a synergizing amount of HMB and a synergizing amount ofleucine, provided that the total amount of HMB and leucine in saidcomposition is less than (or less than about) 3.0 g, wherein: saidsynergizing amount of resveratrol is between at least 35 mg and about500 mg, said synergizing amount of HMB is between about (or at least)0.20 g and about 3.0 g, and said synergizing amount of leucine isbetween about (or at least) 0.50 g and about 3.0 g.

In some embodiments, the composition comprises a synergizing amount ofresveratrol, a synergizing amount of KIC and a synergizing amount ofleucine, provided that the total amount of KIC and leucine in saidcomposition is less than (or less than about) 3.0 g, wherein: saidsynergizing amount of resveratrol is between at least 35 mg and about500 mg, said synergizing amount of KIC is between about (or at least)0.50 g and about 3.0 g, and said synergizing amount of leucine isbetween about (or at least) 0.50 g and about 3.0 g.

In other embodiments, the composition comprises a synergizing amount ofresveratrol, a synergizing amount of HMB and a synergizing amount ofKIC, provided that the total amount of HMB and KIC in said compositionis less than (or less than about) 3.0 g, wherein: said synergizingamount of resveratrol is between at least 35 mg and about 500 mg, saidsynergizing amount of HMB is between about (or at least) 0.20 g andabout 3.0 g, and said synergizing amount of KIC is between about (or atleast) 0.50 g and about 3.0 g.

In some embodiments, the composition comprises a synergizing amount ofresveratrol, a synergizing amount of KIC, a synergizing amount of HMBand a synergizing amount of leucine, provided that the total amount ofKIC, HMB and leucine in said composition is less than (or less thanabout) 3.0 g, wherein: said synergizing amount of resveratrol is betweenat least 35 mg and about 500 mg, said synergizing amount of HMB isbetween about (or at least) 0.20 g and about 3.0 g, said synergizingamount of KIC is between about (or at least) 0.50 g and about 3.0 g, andsaid synergizing amount of leucine is between about (or at least) 0.50 gand about 3.0 g.

In some embodiments, said synergizing amount of resveratrol is betweenat least 50 mg and about 500 mg, and said synergizing amount of HMB isbetween about (or at least) 0.40 g and about 3.0 g.

In some embodiments, said synergizing amount of resveratrol is betweenat least 50 mg and about 500 mg, and said synergizing amount of leucineis between about (or at least) 0.75 g and about 3.0 g.

In some embodiments, said synergizing amount of resveratrol is betweenat least 50 mg and about 500 mg, said synergizing amount of HMB isbetween at least 0.40 g and about 3.0 g, and said synergizing amount ofleucine is between at least 0.75 g and about 3.0 g.

In some embodiments, said synergizing amount of resveratrol is betweenat least 50 mg and about 500 mg, said synergizing amount of KIC isbetween at least 0.75 g and about 3.0 g, and said synergizing amount ofleucine is between at least 0.75 g and about 3.0 g.

In some embodiments, said synergizing amount of resveratrol is betweenat least 50 mg and about 500 mg, said synergizing amount of HMB isbetween at least 0.40 g and about 3.0 g, and said synergizing amount ofKIC is between at least 0.75 g and about 3.0 g.

In some embodiments, said synergizing amount of resveratrol is betweenat least 50 mg and about 500 mg, said synergizing amount of HMB isbetween at least 0.40 g and about 3.0 g, said synergizing amount of KICis between at least 0.75 g and about 3.0 g, and said synergizing amountof leucine is between at least 0.75 g and about 3.0 g.

In some of the embodiments described herein, the amount or HMB, KIC,leucine or combinations of leucine, KIC and/or HMB is less than, orequal to, 3.0 g.

In some of the embodiments described herein, the composition furthercomprises a flavorant. In any one of the embodiments described herein,said composition is a solid, liquid, emulsion, gel or paste. In any oneof the embodiments described herein, the subject is a human or non-humananimal. In any one of the embodiments described herein, said compositionis administered orally, parenterally, intravenously orintraperitoneally. In any of the embodiments for reducing weight gain orinducing weight loss according to any one of embodiments, said subjectis on an unrestricted diet.

In any of the embodiments for reducing weight gain or inducing weightloss according to any one of embodiments, said subject is on a calorierestricted diet. In any one of the embodiments described herein, saidcomposition comprises: a) about 50 to 100 mg resveratrol and about 400mg to about 500 mg HMB, b) about 50 to 100 mg resveratrol and about 750mg to about 1250 mg leucine, or c) about 50 to 100 mg resveratrol andabout 750 mg to about 1250 mg KIC.

In any one of the embodiments described herein, said compositioncomprises about 50 mg to about 100 mg resveratrol and a) a combinationof HMB and KIC in an amount of about 400 mg and about 1250 mg, b) acombination of HMB and leucine in an amount of about 400 mg and about1250 mg, c) a combination of KIC and leucine in an amount of about 400mg and about 1250 mg, or d) a combination of HMB, KIC and leucine in anamount of about 400 mg and about 1250 mg.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawing(s) of which:

FIG. 1 depicts a graph showing the effects of leucine and rapamycin onfatty acid oxidation.

FIG. 2 depicts a graph showing the effects of HMB, KIC, and leucine onfatty acid oxidation.

FIG. 3 depicts a graph showing the effects of HMB, KIC, leucine, andvaline on mitochondrial biogenesis.

FIG. 4 depicts a graph showing the effects of HMB, KIC, and leucine onthe expression of mitochondrial regulatory and component genes.

FIG. 5 depicts a graph showing the effects of resveratrol, suramin,leucine, KIC, HMB, and leucine on SIRT1 activation.

FIG. 6 depicts a graph showing the effects of resveratrol, leucine, HMB,and a combination composition of HMB and Resveratrol on activation ofSirt3.

FIG. 7 depicts a graph showing synergistic effects of leucine and HMBwith resveratrol on fatty acid oxidation under low glucose conditions.

FIG. 8 depicts a graph showing synergistic effects of leucine and HMBwith resveratrol on fatty acid oxidation under high glucose conditions.

FIG. 9 depicts two FDG-PET images showing the synergistic effects ofresveratrol and HMB on glucose uptake using FDG-PET scanning analysis.

FIG. 10 depicts a graph showing the effects of resveratrol, leucine, andHMB on adipose tissue SIRT1 activity in diet-induced obese mice.

FIG. 11 depicts a diagram showing a sirtuin pathway.

FIG. 12 depicts a graph showing interactive effects of chlorogenic acid(500 nM) with HMB (5 μM) and resveratrol (200 nM) on fatty acidoxidation in C2C12 myotubes. Fatty acid oxidation was measured as O₂consumption response to palmitate injection and is expressed as % changefrom pre-injection baseline (vertical line shows the time of palmitateinjection; data points to the left of this line are baselinemeasurements and those to the right of the line show the O₂ consumptionresponse).

FIG. 13 depicts a graph showing interactive effects of chlorogenic acid(500 nM) and HMB (5 μM) on fatty acid oxidation (data expressed as %change from control value. *p=0.05)

FIG. 14 depicts a graph showing interactive effects of chlorogenic acid(500 nM) with HMB (5 μM) and leucine (0.5 mM) on Sirt1 activity in3T3-L1 adipocytes (data expressed as % change from control value;*p=0.005; **p=0.0001).

FIG. 15 depicts a graph showing interactive effects of chlorogenic acid(500 nM) with HMB (5 μM) and leucine (0.5 mM) on glucose utilization(*p=0.045; **p=0.007). Glucose utilization was measured as extracellularacidification response to glucose injection. Response to insulin (5 nM)is included for reference.

FIG. 16 depicts a graph showing interactive effects of caffeic acid (1μM) with leucine (0.5 mM) and resveratrol (200 nM) on fatty acidoxidation in C2C12 myotubes. Fatty acid oxidation was measured as O₂consumption response to palmitate injection and is expressed as % changefrom pre-injection baseline (vertical line shows the time of palmitateinjection; data points to the left of this line are baselinemeasurements and those to the right of the line show the O₂ consumptionresponse).

FIG. 17 depicts a graph showing interactive effects of caffeic acid (1μM) with HMB (5 μM) and resveratrol (200 nM) on fatty acid oxidation inC2C12 myotubes. Fatty acid oxidation was measured as O₂ consumptionresponse to palmitate injection and is expressed as % change frompre-injection baseline (vertical line shows the time of palmitateinjection; data points to the left of this line are baselinemeasurements and those to the right of the line show the O₂ consumptionresponse).

FIG. 18 depicts a graph showing interactive effects of caffeic acid (1μM), HMB (5 leucine (0.5 mM) and resveratrol (200 nM) on fatty acidoxidation in C2C12 myotubes and 3T3-L1 adipocytes (data expressed as %change from control value;*p=0.05; **p=0.016).

FIG. 19 depicts a graph showing interactive effects of quinic acid (500nM) with HMB (5 μM) and resveratrol (200 nM) on fatty acid oxidation in3T3-L1 adipocytes. Fatty acid oxidation was measured as O₂ consumptionresponse to palmitate injection and is expressed as % change frompre-injection baseline (vertical line shows the time of palmitateinjection; data points to the left of this line are baselinemeasurements and those to the right of the line show the O₂ consumptionresponse).

FIG. 20 depicts a graph showing interactive effects of quinic acid (500nM) with leucine (0.5 mM) and resveratrol (200 nM) on fatty acidoxidation in 3T3-L1 adipocytes. Fatty acid oxidation was measured as O₂consumption response to palmitate injection and is expressed as % changefrom pre-injection baseline (vertical line shows the time of palmitateinjection; data points to the left of this line are baselinemeasurements and those to the right of the line show the O₂ consumptionresponse).

FIG. 21 depicts a graph showing interactive effects of quinic acid (500nM), HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on fatty acidoxidation in C2C12 myotubes and 3T3-L1 adipocytes (data expressed as %change from control value; *p=0.05; **p=0.012).

FIG. 22 depicts a graph showing interactive effects of quinic acid (500nM), HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on AMPKactivity (data expressed as % change from control value; *p=0.0001).

FIG. 23 depicts a graph showing interactive effects of quinic acid (500nM), HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on glucoseutilization. Glucose utilization was measured as extracellularacidification response to glucose injection (*p=0.05; **p=0.0003).

FIG. 24 depicts a graph showing interactive effects of cinnamic acid(500 nM) with HMB (5 μM) and resveratrol (200 nM) on fatty acidoxidation in C2C12 myotubes. Fatty acid oxidation was measured as O₂consumption response to palmitate injection and is expressed as % changefrom pre-injection baseline (vertical line shows the time of palmitateinjection; data points to the left of this line are baselinemeasurements and those to the right of the line show the O₂ consumptionresponse).

FIG. 25 depicts a graph showing interactive effects of cinnamic acid(500 nM) with leucine (0.5 mM) and resveratrol (200 nM) on fatty acidoxidation in C2C12 myotubes. Fatty acid oxidation was measured as O₂consumption response to palmitate injection and is expressed as % changefrom pre-injection baseline (vertical line shows the time of palmitateinjection; data points to the left of this line are baselinemeasurements and those to the right of the line show the O₂ consumptionresponse).

FIG. 26 depicts a graph showing interactive effects of cinnamic acid(500 nM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) onfatty acid oxidation in 3T3-L1 adipocytes (data expressed as % changefrom control value; *p=0.004; **p=0.006).

FIG. 27 depicts a graph showing interactive effects of cinnamic acid(500 nM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) onfatty acid oxidation in C2C12 myotubes (data expressed as % change fromcontrol value; *p=0.02; **p=0.05).

FIG. 28 depicts a graph showing interactive effects of cinnamic acid(500 nM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) onAMPK activity (data expressed as % change from control value;*p=0.0001).

FIG. 29 depicts a graph showing interactive effects of ferulic acid (500nM) with HMB (5 μM) and resveratrol (200 nM) on fatty acid oxidation in3T3-L1 adipocytes. Fatty acid oxidation was measured as O₂ consumptionresponse to palmitate injection and is expressed as % change frompre-injection baseline (vertical line shows the time of palmitateinjection; data points to the left of this line are baselinemeasurements and those to the right of the line show the O₂ consumptionresponse).

FIG. 30 depicts a graph showing interactive effects of ferulic acid (500nM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on fattyacid oxidation in 3T3-L1 adipocytes (data expressed as % change fromcontrol value; *p=0.018)

FIG. 31 depicts a graph showing interactive effects of ferulic acid (500nM) with leucine (0.5 mM) and resveratrol (200 nM) on fatty acidoxidation in C2C12 myotubes. Fatty acid oxidation was measured as O₂consumption response to palmitate injection and is expressed as % changefrom pre-injection baseline (vertical line shows the time of palmitateinjection; data points to the left of this line are baselinemeasurements and those to the right of the line show the O₂ consumptionresponse).

FIG. 32 depicts a graph showing interactive effects of ferulic acid (500nM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on fattyacid oxidation in 3T3-L1 adipocytes (data expressed as % change fromcontrol value; *p=0.034).

FIG. 33 depicts a graph showing interactive effects of ferulic acid (500nM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on AMPKactivity (data expressed as % change from control value; *p=0.05).

FIG. 34 depicts a graph showing interactive effects of piceatannol (1nM) with leucine (0.5 mM) and resveratrol (200 nM) on fatty acidoxidation in 3T3-L1 adipocytes. Fatty acid oxidation was measured as O₂consumption response to palmitate injection and is expressed as % changefrom pre-injection baseline (vertical line shows the time of palmitateinjection; data points to the left of this line are baselinemeasurements and those to the right of the line show the O₂ consumptionresponse).

FIG. 35 depicts a graph showing interactive effects of piceatannol (1nM) with HMB (5 μM) and resveratrol (200 nM) on fatty acid oxidation in3T3-L1 adipocytes. Fatty acid oxidation was measured as O₂ consumptionresponse to palmitate injection and is expressed as % change frompre-injection baseline (vertical line shows the time of palmitateinjection; data points to the left of this line are baselinemeasurements and those to the right of the line show the O₂ consumptionresponse).

FIG. 36 depicts a graph showing interactive effects of piceatannol (1nM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on fattyacid oxidation in C2C12 myotubes (data expressed as % change fromcontrol value; *p=0.039).

FIG. 37 depicts a graph showing interactive effects of epigallocatechingallate (EGCG) (1 μM), HMB (5 μM), leucine (0.5 mM) and resveratrol (200nM) on glucose utilization in C2C12 myotubes. Glucose utilization wasmeasured as extracellular acidification response to glucose injection(*p=0.015; **p=0.017).

FIG. 38 depicts a graph showing effects of fucoxanthin (100 nM) with HMB(5 μM) and resveratrol (200 nM) on fatty acid oxidation in 3T3-L1adipocytes. Fatty acid oxidation was measured as O₂ consumption responseto palmitate injection and is expressed as % change from pre-injectionbaseline (vertical line shows the time of palmitate injection; datapoints to the left of this line are baseline measurements and those tothe right of the line show the O₂ consumption response).

FIG. 39 depicts a graph showing interactive effects of fucoxanthin (100nM) leucine (0.5 mM) and resveratrol (200 nM) on fatty acid oxidation in3T3-L1 adipocytes. Fatty acid oxidation was measured as O₂ consumptionresponse to palmitate injection and is expressed as % change frompre-injection baseline (vertical line shows the time of palmitateinjection; data points to the left of this line are baselinemeasurements and those to the right of the line show the O₂ consumptionresponse).

FIG. 40 depicts a graph showing interactive effects of fucoxanthin (100nM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on fattyacid oxidation in 3T3-L1 adipocytes (data expressed as % change fromcontrol value; *p=0.033; **p=0.05).

FIG. 41 depicts a graph showing interactive effects of fucoxanthin (100nM), HMB (5 μM) and leucine (0.5 mM) on glucose utilization in C2C12myotubes. Glucose utilization was measured as extracellularacidification response to glucose injection (*p<0.04).

FIG. 42 depicts a graph showing interactive effects of fucoxanthin (100nM), HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on glucoseutilization in 3T3-L1 adipocytes. Glucose utilization was measured asextracellular acidification response to glucose injection (*p=0.02;**p=0.003).

FIG. 43 depicts a graph showing interactive effects of grape seedextract (1 μg/mL) with HMB (5 μM) and resveratrol (200 nM) on fatty acidoxidation in 3T3-L1 adipocytes. Fatty acid oxidation was measured as O₂consumption response to palmitate injection and is expressed as % changefrom pre-injection baseline (vertical line shows the time of palmitateinjection; data points to the left of this line are baselinemeasurements and those to the right of the line show the O₂ consumptionresponse).

FIG. 44 depicts a graph showing interactive effects of grape seedextract (1 μg/mL) with HMB (5 μM) and resveratrol (200 nM) on fatty acidoxidation in 3T3-L1 adipocytes (data expressed as % change from controlvalue; *p=0.04).

FIG. 45 depicts a graph showing interactive effects of grape seedextract (1 μg/mL) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200nM) on AMPK activity in 3T3-L1 adipocytes and C2C12 myotubes (dataexpressed as % change from control value; *p=0.01).

FIG. 46 depicts a graph showing interactive effects of metformin (0.1mM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on fattyacid oxidation in C2C12 myotubes (data expressed as % change fromcontrol value; *p=0.03; **p=0.0001; ***p=0.001).

FIG. 47 depicts a graph showing interactive effects of metformin (0.1mM) with HMB (5 μM) and leucine (0.5 mM) on glucose utilization in C2C12myotubes. Glucose utilization was measured as extracellularacidification response to glucose injection (*p=0.03).

FIG. 48 depicts a graph showing interactive effects of metformin (0.1mM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on AMPKactivity in C2C12 myotubes (data expressed as % change from controlvalue; *p=0.031; **p=0.026; ***p=0.017)

FIG. 49 depicts a graph showing interactive effects of metformin (0.1mM) with HMB (5 μM) and leucine (0.5 mM) on mitochondrial biogenesis(*p=0.001; **p=0.013).

FIG. 50 depicts a graph showing interactive effects of rosiglitazone (1nM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on fattyacid oxidation in C2C12 myotubes (data expressed as % change fromcontrol value; *p=0.009).

FIG. 51 depicts a graph showing interactive effects of rosiglitazone (1nM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on fattyacid oxidation in 3T3-L1 adipocytes (data expressed as % change fromcontrol value; *p=0.004; **p=0.023; ***p=0.003).

FIG. 52 depicts a graph showing interactive effects of rosiglitazone (1nM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) onglucose utilization in C2C12 myotubes. Glucose utilization was measuredas extracellular acidification response to glucose injection (*p=0.05;**p=0.001).

FIG. 53 depicts a graph showing interactive effects of caffeine (10 nM)with HMB (5 μM), leucine (0.5 mM), resveratrol (200 nM) and metformin(0.1 mM) on fatty acid oxidation in C2C12 myotubes (data expressed as %change from control value; *p=0.03; **p=0.05; ***p=0.013).

FIG. 54 depicts a graph showing interactive effects of caffeine (10 nM)with HMB (5 μM), leucine (0.5 mM), resveratrol (200 nM) and metformin(0.1 mM) on fatty acid oxidation in 3T3-L1 adipocytes (data expressed as% change from control value. *p=0.008).

FIG. 55 depicts a graph showing interactive effects of caffeine (10 nM)with HMB (5 μM) and resveratrol (200 nM) on fatty acid oxidation in3T3-L1 adipocytes. Fatty acid oxidation was measured as O₂ consumptionresponse to palmitate injection and is expressed as % change frompre-injection baseline (vertical line shows the time of palmitateinjection; data points to the left of this line are baselinemeasurements and those to the right of the line show the O₂ consumptionresponse).

FIG. 56 depicts a graph showing interactive effects of theophylline (1μM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on fattyacid oxidation in C2C12 myotubes (data expressed as % change fromcontrol value; *p=0.03; **p=0.05).

FIG. 57 depicts a graph showing interactive effects of theophylline (1μM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on fattyacid oxidation in 3T3-L1 adipocytes. Fatty acid oxidation was measuredas O₂ consumption response to palmitate injection and is expressed as %change from pre-injection baseline (vertical line shows the time ofpalmitate injection; data points to the left of this line are baselinemeasurements and those to the right of the line show the O₂ consumptionresponse).

FIG. 58 depicts a graph showing interactive effects of theophylline (1μM) with HMB (5 μM), leucine (0.5 mM) and resveratrol (200 nM) on fattyacid oxidation in 3T3-L1 adipocytes (data expressed as % change fromcontrol value; *p=0.006).

FIG. 59 depicts a graph showing interactive effects of cocoaextract/theobromine (0.1 μg/mL) with HMB (5 μM), leucine (0.5 mM) andresveratrol (200 nM) on fatty acid oxidation in 3T3-L1 adipocytes. Fattyacid oxidation was measured as O₂ consumption response to palmitateinjection and is expressed as % change from pre-injection baseline(vertical line shows the time of palmitate injection; data points to theleft of this line are baseline measurements and those to the right ofthe line show the O₂ consumption response).

FIG. 60 depicts a graph showing interactive effects of cocoaextract/theobromine (0.1 μg/mL) with HMB (5 μM), leucine (0.5 mM) andresveratrol (200 nM) on fatty acid oxidation in 3T3-L1 adipocytes (dataexpressed as % change from control value; *p=0.021; **p=0.00035).

FIG. 61 depicts a graph showing effects of a standard dose of metformin(here 1.5 g metformin/kg diet), a low dose of metformin (here 0.75 gmetformin/kg diet) and a very lose dose of metformin (here 0.25 gmetformin/kg diet) compared with [the low dose of metformin+12.5 mgresveratrol and 2 g CaHMB/kg diet] and with [the very lose dose ofmetformin+12.5 mg resveratrol and 2 g CaHMB/kg diet] on plasma insulinin db/db mice (*p<0.02 vs. control).

FIG. 62 depicts a graph showing effects of a standard dose of metformin(here 1.5 g metformin/kg diet), a low dose of metformin (here 0.75 gmetformin/kg diet) and a very lose dose of metformin (here 0.25 gmetformin/kg diet) compared with [the low dose of metformin+12.5 mgresveratrol and 2 g CaHMB/kg diet] and with [the very lose dose ofmetformin+12.5 mg resveratrol and 2 g CaHMB/kg diet] on HOMA_(IR)(homeostatic assessment of insulin resistance) in db/db mice (*p<0.025vs. control).

FIG. 63 depicts a graph showing effects of a standard dose of metformin(here 1.5 g metformin/kg diet), a low dose of metformin (here 0.75 gmetformin/kg diet) and a very lose dose of metformin (here 0.25 gmetformin/kg diet) compared with [the low dose of metformin+12.5 mgresveratrol and 2 g CaHMB/kg diet] and with [the very lose dose ofmetformin+12.5 mg resveratrol and 2 g CaHMB/kg diet] on 30-minute plasmaglucose response to insulin (0.75 U/kg body weight) in db/db mice(*p<0.02 vs. control).

FIG. 64 depicts a graph showing effects of a standard dose of metformin(here 1.5 g metformin/kg diet), a low dose of metformin (here 0.75 gmetformin/kg diet) and a very lose dose of metformin (here 0.25 gmetformin/kg diet) compared with [the low dose of metformin+12.5 mgresveratrol and 2 g CaHMB/kg diet] and with [the very lose dose ofmetformin+12.5 mg resveratrol and 2 g CaHMB/kg diet] on visceral fatmass in db/db mice (*p<0.03 vs. control).

FIG. 65 depicts a graph showing effects of a standard dose of metformin(here 1.5 g metformin/kg diet), a low dose of metformin (here 0.75 gmetformin/kg diet) and a very lose dose of metformin (here 0.25 gmetformin/kg diet) compared with [the low dose of metformin+12.5 mgresveratrol and 2 g CaHMB/kg diet] and with [the very lose dose ofmetformin+12.5 mg resveratrol and 2 g CaHMB/kg diet] on visceral fatmass in db/db mice (*p<0.05 vs. control).

FIG. 66 depicts a graph showing interactive effects of resveratrol (200nM), leucine (0.5 mM), HMB (5 μM) and cinnamic acid (1 μM) on fatty acidoxidation (*p=0.035 vs. control).

FIG. 67 depicts a graph showing interactive effects of resveratrol (200nM), leucine (0.5 mM), HMB (5 μM) and cinnamic acid (1 μM) on expressionof irisin precursor protein FNDC5 in C2C12 cellular lysates measured byWestern blot (values are normalized band intensity units; *p<0.03 vs.control).

FIG. 68 depicts a representative Western blot of irisin secretion intoculture media in response to leucine (0.5 mM) or HMB (5 μM) combinedwith either resveratrol (200 nM) or cinnamic acid (1 μM). Quantitativedata normalized to control: resveratrol/HMB: 73% increase (p<0.01);resveratrol/leucine, 271% increase (p<0.01), cinnamic acid/HMB 7% (notsignificant), cinnamic acid/leucine, 238% (p<0.01).

FIG. 69 depicts a graph showing interactive effects of resveratrol (200nM), leucine (0.5 mM) and HMB (5 μM) on irisin secretion by C2C12myotubes into culture media (*p=0.0008 vs. control; **p=0.00001 vs.control).

FIG. 70 depicts a graph showing interactive effects of resveratrol andresveratrol/leucine combination on plasma irisin in diet-induced obesemice (*p=0.03 vs. control).

FIG. 71 depicts a graph showing interactive effects of quinic acid (QA;500 nM), leucine (Leu; 0.5 mM), HMB (5 μM) on FNDC5 protein expressionin C2C12 cellular lysates measured by Western blot. Values arenormalized band intensity units (*p<0.005 vs. control).

FIG. 72 depicts a representative Western blot of irisin secretion byC2C12 cells into the culture media in response to leucine (0.5 mM) orHMB (5 μM) combined with quinic acid (500 nM).

FIG. 73 depicts a graph showing quantitative assessment of the effectsof leucine (0.5 mM) or HMB (5 μM) combined with quinic acid (500 nM) onirisin secretion by C2C12 cells into culture media (*p<0.05 vs.control).

FIG. 74 depicts a graph showing interactive effects of chlorogenic acid(CA; 500 nM), caffeine (Cafn; 10 nM), leucine (Leu; 0.5 mM), and HMB (5μM) on irisin secretion by C2C12 myotubes into culture media (*p<0.05 vscontrol).

FIG. 75 depicts a graph showing the effects of resveratrol,resveratrol/leucine, and resveratrol/HMB combination on subcutaneousUCP1 mRNA expression in diet-induced obese mice. Data are normalized to18S (*p=0.05 vs. control).

FIG. 76 depicts a graph showing effects of resveratrol,resveratrol/leucine, and resveratrol/HMB combination on subcutaneousPGC1α mRNA expression in diet-induced obese mice. Data are normalized to18S (*p=0.04 vs. control).

DETAILED DESCRIPTION OF THE INVENTION

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. Unless stated otherwise, the present invention is notlimited by the illustrated ordering of acts or events, as some acts mayoccur in different orders and/or concurrently with other acts or events.Furthermore, not all illustrated acts or events are required toimplement a methodology in accordance with the present invention. Theconcentration of various components in the disclosed compositions areexemplary and not meant to be limited to the recited concentration perse.

As used herein, the term “subject” or “individual” includes mammals.Non-limiting examples of mammals include humans and mice, includingtransgenic and non-transgenic mice. The methods described herein can beuseful in both human therapeutics, pre-clinical, and veterinaryapplications. In some embodiments, the subject is a mammal, and in someembodiments, the subject is human. Other mammals include, and are notlimited to, apes, chimpanzees, orangutans, monkeys; domesticated animals(pets) such as dogs, cats, guinea pigs, hamsters, mice, rats, rabbits,and ferrets; domesticated farm animals such as cows, buffalo, bison,horses, donkey, swine, sheep, and goats; or exotic animals typicallyfound in zoos, such as bear, lions, tigers, panthers, elephants,hippopotamus, rhinoceros, giraffes, antelopes, sloth, gazelles, zebras,wildebeests, prairie dogs, koala bears, kangaroo, pandas, giant pandas,hyena, seals, sea lions, and elephant seals.

The terms “administer”, “administered”, “administers” and“administering” are defined as the providing a composition to a subjectvia a route known in the art, including but not limited to intravenous,intraarterial, oral, parenteral, buccal, topical, transdermal, rectal,intramuscular, subcutaneous, intraosseous, transmucosal, orintraperitoneal routes of administration. In certain embodiments of thesubject application, oral routes of administering a composition may bepreferred.

As used herein, “agent” or “biologically active agent” refers to abiological, pharmaceutical, or chemical compound or other moiety.Non-limiting examples include simple or complex organic or inorganicmolecule, a peptide, a protein, a peptide nucleic acid (PNA), anoligonucleotide (including e.g., aptomer and polynucleotides), anantibody, an antibody derivative, antibody fragment, a vitaminderivative, a carbohydrate, a toxin, or a chemotherapeutic compound.Various compounds can be synthesized, for example, small molecules andoligomers (e.g., oligopeptides and oligonucleotides), and syntheticorganic compounds based on various core structures. In addition, variousnatural sources can provide compounds for screening, such as plant oranimal extracts, and the like. A skilled artisan can readily recognizethat there is no limit as to the structural nature of the agents of thepresent invention.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound described herein that is sufficient toeffect the intended application including but not limited to diseasetreatment, as defined below. The therapeutically effective amount mayvary depending upon the intended application (in vitro or in vivo), orthe subject and disease condition being treated, e.g., the weight andage of the subject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art. The term also applies to a dose that willinduce a particular response in target cells, e.g., reduction ofproliferation or down regulation of activity of a target protein. Thespecific dose will vary depending on the particular compounds chosen,the dosing regimen to be followed, whether it is administered incombination with other compounds, timing of administration, the tissueto which it is administered, and the physical delivery system in whichit is carried.

A “modulator” of a pathway refers to a substance or agent whichmodulates the activity of one or more cellular proteins mapped to thesame specific signal transduction pathway. A modulator may augment orsuppress the activity and/or expression level or pattern of a signalingmolecule. A modulator can activate a component in a pathway by directlybinding to the component. A modulator can also indirectly activate acomponent in a pathway by interacting with one or more associatedcomponents. The output of the pathway can be measured in terms of theexpression or activity level of proteins. The expression level of aprotein in a pathway can be reflected by levels of corresponding mRNA orrelated transcription factors as well as the level of the protein in asubcellular location. For instance, certain proteins are activated bytranslocating in or out of a specific subcellular component, includingbut not limited to nucleus, mitochondria, endosome, lysosome or othermembraneous structure of a cell. The output of the pathway can also bemeasured in terms of physiological effects, such as mitochondrialbiogenesis, fatty acid oxidation, or glucose uptake.

An “activator” refers to a modulator that influences a pathway in amanner that increases the pathway output. Activation of a particulartarget may be direct (e.g. by interaction with the target) or indirect(e.g. by interaction with a protein upstream of the target in asignaling pathway including the target).

A “suppressor” can be a modulator that influences a pathway in a mannerthat decreases pathway output.

The term “substantially free”, as used herein, refers to compositionsthat have less than about 10%, less than about 5%, less than about 1%,less than about 0.5%, less than 0.1% or even less of a specifiedcomponent. For example a composition that is substantially free ofnon-branched chain amino acids may have less than about 1% of thenon-branched chain amino acid lysine.

A “sub-therapeutic amount” of an agent, an activator or a therapy is anamount less than the effective amount for that agent, activator ortherapy, but when combined with an effective or sub-therapeutic amountof another agent or therapy can produce a desired result, due to, forexample, synergy in the resulting efficacious effects, and/or reducedside effects.

A “synergistic” or “synergizing” effect can be such that the one or moreeffects of the combination compositions are greater than the one or moreeffects of each component alone, or they can be greater than the sum ofthe one or more effects of each component alone. The synergistic effectcan be about, or greater than about 10, 20, 30, 50, 75, 100, 110, 120,150, 200, 250, 350, or 500% or even more than the effect on a subjectwith one of the components alone, or the additive effects of each of thecomponents when administered individually. The effect can be any of themeasurable effects described herein.

Compositions

The invention provides for compositions that can increase or modulatethe output of a sirtuin pathway. The sirtuin pathway includes, withoutlimitation, signaling molecules such as, Sirt1, Sirt3, and AMPK. Theoutput of the pathway can be determined by the expression level and/orthe activity of the pathway and/or a physiological effect. In someembodiments, activation of the Sirt1 pathway includes stimulation ofPGC1-α and/or subsequent stimulation of mitochondrial biogenesis andfatty acid oxidation. In general, a sirtuin pathway activator iscompound that activates or increases one or more components of a sirtuinpathway. An increase or activation of a sirtuin pathway can be observedby an increase in the activity of a pathway component protein. Forexample, the protein can be Sirt1, PGC1-α, AMPK, Epac1, Adenylylcyclase, Sirt3, or any other proteins and their respective associatedproteins along the signaling pathway depicted in FIG. 11 (Park et. al.,“Resveratrol Ameliorates Aging-Related Metabolic Phenotypes byInhibiting cAMP Phosphodiesterases,” Cell 148, 421-433 Feb. 3, 2012).Non-limiting examples of physiological effects that can serve asmeasures of sirtuin pathway output include mitochondrial biogenesis,fatty acid oxidation, glucose uptake, palmitate uptake, oxygenconsumption, carbon dioxide production, weight loss, heat production,visceral adipose tissue loss, respiratory exchanger ratio, insulinsensitivity, inflammation marker level, vasodilation, browing of fatcells, and irisin production. Examples of indicia of browning of fatcells include, without limitation, increased fatty acid oxidation, andexpression of one or more brown-fat-selective genes (e.g. Ucp1, Cidea,Prdm16, and Ndufs1). In some embodiments, changes in one or morephysiological effects that can serve as measures of sirtuin pathwayoutput are induced by increasing irisin production, such as byadministering a composition of the invention.

An increase in mitochondrial biogenesis can be evidenced by an increasein the formation of new mitochondria and/or by an increase inmitochondrial functions, such as increased fatty acid oxidation,increased heat generation, increased insulin sensitivity, increased inglucose uptake, increased in vasodilation, decreased in weight,decreased in adipose volume, and decreased inflammatory response ormarkers in a subject.

The compositions can be combination compositions which may include oneor more synergistic components. In some embodiments, the synergisticeffect of the combination compositions can allow for reduced dosingamounts, leading to reduced side effects to the subject and reduced costof treatment. In other embodiments, the synergistic effect can allow forresults that are not achievable through any other conventionaltreatments. The subject combination compositions provide a significantimprovement in the regulation of energy metabolism.

In some embodiments, the compositions can be combination compositions ofone or more branched chain amino acids and/or metabolites thereof and asirtuin-pathway activator can have one or more characteristics. Thecombination compositions (a) can have a synergistic effect in increasingthe sirtuin-pathway output, (b) increase sirtuin-pathway output by atleast about 1, 2, 5, 7, 10, or 20 fold, (c) have a molar ratio ofbranched chain amino acids and/or metabolites thereof to sirtuin-pathwayoutput that is greater than about 20, (d) be formulated as a unit dosagefor oral ingestion, where the sirtuin-pathway activator is asubstantially homogeneous population of polyphenol molecules, and (e)can have a synergistic effect and further comprise a food carrier. Anyof the compositions described herein can have one or more of thesecharacteristics.

In some embodiments, the present invention provides a compositioncomprising (a) one or more types of branched amino acids and/ormetabolites thereof and (b) a sirtuin-pathway activator present in asub-therapeutic amount, wherein the composition is synergisticallyeffective in increasing the sirtuin-pathway output by at least about 5,10, 50, 100, 200, 500 or more fold as compared to that of component (b)when it being used alone.

In some embodiments, the sirtuin-pathway activator or AMPK pathwayactivator can be a polyphenol. For example, the polyphenol can bechlorogenic acid, resveratrol, caffeic acid, piceatannol, ellagic acid,epigallocatechin gallate (EGCG), grape seed extract, or any analogthereof. In some embodiments, the activator can be resveratrol, ananalog thereof, or a metabolite thereof. For example, the activator canbe pterostilbene or a small molecule analog of resveratrol. Examples ofsmall molecule analogs of resveratrol are described in U.S. PatentApplication Nos. 20070014833, 20090163476, and 20090105246, which areincorporated herein by reference in its entirety.

The polyphenol can be a substantially homogeneous population ofpolyphenols. The polyphenol can be one type of polyphenol, wherein thecomposition can exclude all other types of polyphenols. In otherembodiments, the composition can comprise two, three, or four types ofpolyphenols, and exclude all other types of polyphenols. In someembodiments, the composition can comprise 1, 2, 3, or 4 types ofpolyphenols and less than 0.1, 0.5, 1, or 2% of any other types ofpolyphenols. In some embodiments, a composition further comprises aphosphodiesterase (PDE) inhibitor, and/or other sirtuin pathwayactivator.

In some embodiments, a sirtuin activator is any one or more of thecompounds shown below:

In one embodiment, a sirtuin activator is a stilbene or chalconecompound of formula 1:

wherein, independently for each occurrence,

R₁, R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, and R′₅ represent H, alkyl,aryl, heteroaryl, aralkyl, alkaryl, heteroaralkyl, halide, NO₂, SR, OR,N(R)₂, or carboxyl;

R represents H, alkyl, aryl, heteroaryl, or aralkyl;

M represents O, NR, or S;

A-B represents a bivalent alkyl, alkenyl, alkynyl, amido, sulfonamido,diazo, ether, alkylamino, alkylsulfide, hydroxylamine, or hydrazinegroup; and

n is 0 or 1.

In a further embodiment, a sirtuin activator is a compound of formula 1and the attendant definitions, wherein n is 0. In a further embodiment,a sirtuin activator is a compound of formula 1 and the attendantdefinitions, wherein n is 1. In a further embodiment, a sirtuinactivator is a compound of formula 1 and the attendant definitions,wherein A-B is ethenyl. In a further embodiment, a sirtuin activator isa compound of formula 1 and the attendant definitions, wherein A-B is—CH₂CH(Me)CH(Me)CH₂—. In a further embodiment, a sirtuin activator is acompound of formula 1 and the attendant definitions, wherein M is O. Ina further embodiment, the methods comprises a compound of formula 1 andthe attendant definitions, wherein R₁, R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃,R′₄, and R′₅ are H. In a further embodiment, a sirtuin activator is acompound of formula 1 and the attendant definitions, wherein R₂, R₄, andR′₃ are OH. In a further embodiment, a sirtuin activator is a compoundof formula 1 and the attendant definitions, wherein R₂, R₄, R′₂ and R′₃are OH. In a further embodiment, a sirtuin activator is a compound offormula 1 and the attendant definitions, wherein R₃, R₅, R′₂ and R′₃ areOH. In a further embodiment, a sirtuin activator is a compound offormula 1 and the attendant definitions, wherein R₁, R₃, R₅, R′₂ and R′₃are OH. In a further embodiment, a sirtuin activator is a compound offormula 1 and the attendant definitions, wherein R₂ and R′₂ are OH; R₄is O-β-D-glucoside; and R′₃ is OCH₃. In a further embodiment, a sirtuinactivator is a compound of formula 1 and the attendant definitions,wherein R₂ is OH; R₄ is O-β-D-glucoside; and R′₃ is OCH₃.

In a further embodiment, a sirtuin activator is a compound of formula 1and the attendant definitions, wherein n is 0; A-B is ethenyl; and R₁,R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, and R′₅ are H (trans stilbene). In afurther embodiment, a sirtuin activator is a compound of formula 1 andthe attendant definitions, wherein n is 1; A-B is ethenyl; M is O; andR₁, R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, and R′₅ are H (chalcone). In afurther embodiment, a sirtuin activator is a compound of formula 1 andthe attendant definitions, wherein n is 0; A-B is ethenyl; R₂, R₄, andR′₃ are OH; and R₁, R₃, R₅, R′₁, R′₂, R′₄, and R′₅ are H (resveratrol).In a further embodiment, a sirtuin activator is a compound of formula 1and the attendant definitions, wherein n is 0; A-B is ethenyl; R₂, R₄,R′₂ and R′₃ are OH; and R₁, R₃, R₅, R′₁, R′₄ and R′₅ are H(piceatannol). In a further embodiment, a sirtuin activator is acompound of formula 1 and the attendant definitions, wherein n is 1; A-Bis ethenyl; M is O; R₃, R₅, R′₂ and R′₃ are OH; and R₁, R₂, R₄, R′₁,R′₄, and R′₅ are H (butein). In a further embodiment, a sirtuinactivator is a compound of formula 1 and the attendant definitions,wherein n is 1; A-B is ethenyl; M is O; R₁, R₃, R₅, R′₂ and R′₃ are OH;and R₂, R₄, R′₁, R′₄, and R′₅ are H (3,4,2′,4′,6′-pentahydroxychalcone).In a further embodiment, a sirtuin activator is a compound of formula 1and the attendant definitions, wherein n is 0; A-B is ethenyl; R₂ andR′₂ are OH, R₄ is O-β-D-glucoside, R′₃ is OCH₃; and R₁, R₃, R₅, R′₁,R′₄, and R′₅ are H (rhapontin). In a further embodiment, a sirtuinactivator is a compound of formula 1 and the attendant definitions,wherein n is 0; A-B is ethenyl; R₂ is OH, R₄ is O-β-D-glucoside, R′₃ isOCH₃; and R₁, R₃, R₅, R′₁, R′₂, R′₄, and R′₅ are H (deoxyrhapontin). Ina further embodiment, a sirtuin activator is a compound of formula 1 andthe attendant definitions, wherein n is 0; A-B is —CH₂CH(Me)CH(Me)CH₂—;R₂, R₃, R′₂, and R′₃ are OH; and R₁, R₄, R₅, R′₁, R′₄, and R′₅ are H(NDGA).

In another embodiment, a sirtuin activator is a flavanone compound offormula 2:

wherein, independently for each occurrence,

R₁, R₂, R₃, R₄, R′₁, R′₂, R′₃, R′₄, R′₅, and R″ represent H, alkyl,aryl, heteroaryl, aralkyl, alkaryl, heteroaralkyl, halide, NO₂, SR, OR,N(R)₂, or carboxyl;

R represents H, alkyl, aryl, heteroaryl, or aralkyl;

M represents H₂, O, NR, or S;

Z represents CR, O, NR, or S;

X represents CR or N; and

Y represents CR or N.

In a further embodiment, a sirtuin activator is a compound of formula 2and the attendant definitions, wherein X and Y are both CH. In a furtherembodiment, a sirtuin activator is a compound of formula 2 and theattendant definitions, wherein M is O. In a further embodiment, asirtuin activator is a compound of formula 2 and the attendantdefinitions, wherein M is H₂. In a further embodiment, a sirtuinactivator is a compound of formula 2 and the attendant definitions,wherein Z is O. In a further embodiment, a sirtuin activator is acompound of formula 2 and the attendant definitions, wherein R″ is H. Ina further embodiment, a sirtuin activator is a compound of formula 2 andthe attendant definitions, wherein R″ is OH. In a further embodiment, asirtuin activator is a compound of formula 2 and the attendantdefinitions, wherein R″ is an alkoxycarbonyl. In a further embodiment, asirtuin activator is a compound of formula 2 and the attendantdefinitions, wherein R₁ is

In a further embodiment, a sirtuin activator is a compound of formula 2and the attendant definitions, wherein R₁, R₂, R₃, R₄, R′₁, R′₂, R′₃,R′₄, R′₅ and R″ are H. In a further embodiment, a sirtuin activator is acompound of formula 2 and the attendant definitions, wherein R₂, R₄, andR′₃ are OH. In a further embodiment, a sirtuin activator is a compoundof formula 2 and the attendant definitions, wherein R₄, R′₂, R′₃, and R″are OH. In a further embodiment, a sirtuin activator is a compound offormula 2 and the attendant definitions, wherein R₂, R₄, R′₂, R′₃, andR″ are OH. In a further embodiment, a sirtuin activator is a compound offormula 2 and the attendant definitions, wherein R₂, R₄, R′₂, R′₃, R′₄,and R″ are OH.

In a further embodiment, a sirtuin activator is a compound of formula 2and the attendant definitions, wherein X and Y are CH; M is O; Z and O;R″ is H; and R₁, R₂, R₃, R₄, R′₁, R′₂, R′₃, R′₄, R′₅ and R″ are H(flavanone). In a further embodiment, a sirtuin activator is a compoundof formula 2 and the attendant definitions, wherein X and Y are CH; M isO; Z and O; R″ is H; R₂, R₄, and R′₃ are OH; and R₁, R₃, R′₁, R′₂, R′₄,and R′₅ are H (naringenin). In a further embodiment, a sirtuin activatoris a compound of formula 2 and the attendant definitions, wherein X andY are CH; M is O; Z and O; R″ is OH; R₂, R₄, R′₂, and R′₃ are OH; andR′₁, R₃, R′₁, R′₄, and R′₅ are H (3,5,7,3′,4′-pentahydroxyflavanone). Ina further embodiment, a sirtuin activator is a compound of formula 2 andthe attendant definitions, wherein X and Y are CH; M is H₂; Z and O; R″is OH; R₂, R₄, R′₂, and R′₃, are OH; and R₁, R₃, R′₁, R′₄ and R′₅ are H(epicatechin). In a further embodiment, a sirtuin activator is acompound of formula 2 and the attendant definitions, wherein X and Y areCH; M is H₂; Z and O; R″ is OH; R₂, R₄, R′₂, R′₃, and R′₄ are OH; andR₁, R₃, R′₁, and R′₅ are H (gallocatechin). In a further embodiment, asirtuin activator is a compound of formula 2 and the attendantdefinitions, wherein X and Y are CH; M is H₂; Z and O; R″ is

R₂, R₄, R′₂, R′₃, R′₄, and R″ are OH; and R₁, R₃, R′₁, and R′₅ are H(epigallocatechin gallate).

In another embodiment, a sirtuin activator is an isoflavanone compoundof formula 3:

wherein, independently for each occurrence,

R₁, R₂, R₃, R₄, R′₁, R′₂, R′₃, R′₄, R′₅, and R″₁ represent H, alkyl,aryl, heteroaryl, aralkyl, alkaryl, heteroaralkyl, halide, NO₂, SR, OR,N(R)₂, or carboxyl;

R represents H, alkyl, aryl, heteroaryl, or aralkyl;

M represents H₂, O, NR, or S;

Z represents C(R)₂, O, NR, or S;

X represents CR or N; and

Y represents CR or N.

In another embodiment, a sirtuin activator is a flavone compound offormula 4:

wherein, independently for each occurrence,

R₁, R₂, R₃, R₄, R′₁, R′₂, R′₃, R′₄, and R′₅, represent H, alkyl, aryl,heteroaryl, aralkyl, alkaryl, heteroaralkyl, halide, NO₂, SR, OR, N(R)₂,or carboxyl;

R represents H, alkyl, aryl, heteroaryl, or aralkyl;

M represents H₂, O, NR, or S;

Z represents CR, O, NR, or S; and

X represents CR″ or N, wherein

R″ is H, alkyl, aryl, heteroaryl, alkaryl, heteroaralkyl, halide, NO₂,SR, OR, N(R)₂, or carboxyl.

In a further embodiment, a sirtuin activator is a compound of formula 4and the attendant definitions, wherein X is C. In a further embodiment,a sirtuin activator is a compound of formula 4 and the attendantdefinitions, wherein X is CR. In a further embodiment, a sirtuinactivator is a compound of formula 4 and the attendant definitions,wherein Z is O. In a further embodiment, a sirtuin activator is acompound of formula 4 and the attendant definitions, wherein M is O. Ina further embodiment, a sirtuin activator is a compound of formula 4 andthe attendant definitions, wherein R″ is H. In a further embodiment, asirtuin activator is a compound of formula 4 and the attendantdefinitions, wherein R″ is OH. In a further embodiment, a sirtuinactivator is a compound of formula 4 and the attendant definitions,wherein R₁, R₂, R₃, R₄, R′₁, R′₂, R′₃, R′₄, and R′₅ are H. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein R₂, R′₂, and R′₃ are OH. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein R₂, R₄, R′₂, R′₃, and R′₄ are OH. In afurther embodiment, a sirtuin activator is a compound of formula 4 andthe attendant definitions, wherein R₂, R₄, R′₂, and R′₃ are OH. In afurther embodiment, a sirtuin activator is a compound of formula 4 andthe attendant definitions, wherein R₃, R′₂, and R′₃ are OH. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein R₂, R₄, R′₂, and R′₃ are OH. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein R₂, R′₂, R′₃, and R′₄ are OH. In afurther embodiment, a sirtuin activator is a compound of formula 4 andthe attendant definitions, wherein R₂, R₄, and R′₃ are OH. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein R₂, R₃, R₄, and R′₃ are OH. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein R₂, R₄, and R′₃ are OH. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein R₃, R′₁, and R′₃ are OH. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein R₂ and R′₃ are OH. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein R₁, R₂, R′₂, and R′₃ are OH. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein R₃, R′₁, and R′₂ are OH. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein R′₃ is OH. In a further embodiment, asirtuin activator is a compound of formula 4 and the attendantdefinitions, wherein R₄ and R′₃ are OH. In a further embodiment, asirtuin activator is a compound of formula 4 and the attendantdefinitions, wherein R₂ and R₄ are OH. In a further embodiment, asirtuin activator is a compound of formula 4 and the attendantdefinitions, wherein R₂, R₄, R′₁, and R′₃ are OH. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein R₄ is OH. In a further embodiment, asirtuin activator is a compound of formula 4 and the attendantdefinitions, wherein R₂, R₄, R′₂, R′₃, and R′₄ are OH. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein R₂, R′₂, R′₃, and R′₄ are OH. In afurther embodiment, a sirtuin activator is a compound of formula 4 andthe attendant definitions, wherein R₁, R₂, R₄, R′₂, and R′₃ are OH.

In a further embodiment, a sirtuin activator is a compound of formula 4and the attendant definitions, wherein X is CH; Z is O; M is O; and R₁,R₂, R₃, R₄, R′₁, R′₂, R′₃, R′₄, and R′₅ are H (flavone). In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein X is COH; Z is O; M is O; R₂, R′₂, andR′₃ are OH; and R₁, R₃, R₄, R′₁, R′₄, and R′₅ are H (fisetin). In afurther embodiment, a sirtuin activator is a compound of formula 4 andthe attendant definitions, wherein X is CH; Z is O; M is O; R₂, R₄, R′₂,R′₃, and R′₄ are OH; and R₁, R₃, R′₁, and R′₅ are H(5,7,3′,4′,5′-pentahydroxyflavone). In a further embodiment, a sirtuinactivator is a compound of formula 4 and the attendant definitions,wherein X is CH; Z is O; M is O; R₂, R₄, R′₂, and R′₃ are OH; and R₁,R₃, R′₁, R′₄, and R′₅ are H (luteolin). In a further embodiment, asirtuin activator is a compound of formula 4 and the attendantdefinitions, wherein X is COH; Z is O; M is O; R₃, R′₂, and R′₃ are OH;and R₁, R₂, R₄, R′₁, R′₄, and R′₅ are H (3,6,3′,4′-tetrahydroxyflavone).In a further embodiment, a sirtuin activator is a compound of formula 4and the attendant definitions, wherein X is COH; Z is O; M is O; R₂, R₄,R′₂, and R′₃ are OH; and R₁, R₃, R′₁, R′₄, and R′₅, are H (quercetin).In a further embodiment, a sirtuin activator is a compound of formula 4and the attendant definitions, wherein X is CH; Z is O; M is O; R₂, R′₂,R′₃, and R′₄ are OH; and R₁, R₃, R₄, R′₁, and R′₅ are H. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein X is COH; Z is O; M is O; R₂, R₄, and R′₃are OH; and R₁, R₃, R′₁, R′₂, R′₄, and R′₅ are H. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein X is CH; Z is O; M is O; R₂, R₃, R₄, andR′₃ are OH; and R₁, R′₁, R′₂, R′₄, and R′₅ are further embodiment, asirtuin activator is a compound of formula 4 and the attendantdefinitions, wherein X is CH; Z is O; M is O; R₂, R₄, and R′₃ are OH;and R₁, R₃, R′₁, R′₂, R′₄, and R′₅ are H. In a further embodiment, asirtuin activator is a compound of formula 4 and the attendantdefinitions, wherein X is COH; Z is O; M is O; R₃, R′₁, and R′₃ are OH;and R₁, R₂, R₄, R′₂, R′₄, and R′₅ are H. In a further embodiment, asirtuin activator is a compound of formula 4 and the attendantdefinitions, wherein X is CH; Z is O; M is O; R₂ and R′₃ are OH; and R₁,R₃, R₄, R′₁, R′₂, R′₄, and R′₅ are H. In a further embodiment, a sirtuinactivator is a compound of formula 4 and the attendant definitions,wherein X is COH; Z is O; M is O; R₁, R₂, R′₂, and R′₃ are OH; and R₁,R₂, R₄, R′₃, R′₄, and R′₅ are H. In a further embodiment, a sirtuinactivator is a compound of formula 4 and the attendant definitions,wherein X is COH; Z is O; M is O; R₃, R′₁, and R′₂ are OH; and R₁, R₂,R₄; R′₃, R′₄, and R′₅ are H. In a further embodiment, a sirtuinactivator is a compound of formula 4 and the attendant definitions,wherein X is CH; Z is O; M is O; R′₃ is OH; and R₁, R₂, R₃, R₄, R′₁, R₂,R′₄, and R′₅ are H. In a further embodiment, a sirtuin activator is acompound of formula 4 and the attendant definitions, wherein X is CH; Zis O; M is O; R₄ and R′₃ are OH; and R₁, R₂, R₃, R′₁, R′₂, R′₄, and R′₅are H. In a further embodiment, a sirtuin activator is a compound offormula 4 and the attendant definitions, wherein X is CH; Z is O; M isO; R₂ and R₄ are OH; and R₁, R₃, R′₁, R′₂, R′₃, R′₄, and R′₅ are H. In afurther embodiment, a sirtuin activator is a compound of formula 4 andthe attendant definitions, wherein X is COH; Z is O; M is O; R₂, R₄,R′₁, and R′₃ are OH; and R₁, R₃, R′₂, R′₄, and R′₅ are H. In a furtherembodiment, a sirtuin activator is a compound of formula 4 and theattendant definitions, wherein X is CH; Z is O; M is O; R₄ is OH; andR₁, R₂, R₃, R′₁, R′₂, R′₃, R′₄, and R′₅ are H. In a further embodiment,a sirtuin activator is a compound of formula 4 and the attendantdefinitions, wherein X is COH; Z is O; M is O; R₂, R₄, R′₂, R′₃, and R′₄are OH; and R₁, R₃, R′₁, and R′₅, are H. In a further embodiment, asirtuin activator is a compound of formula 4 and the attendantdefinitions, wherein X is COH; Z is O; M is O; R₂, R′₂, R′₃, and R′₄ areOH; and R₁, R₃, R₄, R′₁, and R′₅ are H. In a further embodiment, asirtuin activator is a compound of formula 4 and the attendantdefinitions, wherein X is COH; Z is O; M is O; R₁, R₂, R₄, R′₂, and R′₃are OH; and R₃, R′₁, R′₄, and R′₅ are H.

In another embodiment, a sirtuin activator is an isoflavone compound offormula 5:

wherein, independently for each occurrence,

R₁, R₂, R₃, R₄, R′₁, R′₂, R′₃, R′₄, and R′₅, represent H, alkyl, aryl,heteroaryl, aralkyl, alkaryl, heteroaralkyl, halide, NO₂, SR, OR, N(R)₂,or carboxyl;

R represents H, alkyl, aryl, heteroaryl, or aralkyl;

M represents H₂, O, NR, or S;

Z represents C(R)₂, O, NR, or S; and

Y represents CR″ or N, wherein

R″ represents H, alkyl, aryl, heteroaryl, alkaryl, heteroaralkyl,halide, NO₂, SR, OR, N(R)₂, or carboxyl.

In a further embodiment, a sirtuin activator is a compound of formula 5and the attendant definitions, wherein Y is CR″. In a furtherembodiment, a sirtuin activator is a compound of formula 5 and theattendant definitions, wherein Y is CH. In a further embodiment, asirtuin activator is a compound of formula 5 and the attendantdefinitions, wherein Z is O. In a further embodiment, a sirtuinactivator is a compound of formula 5 and the attendant definitions,wherein M is O. In a further embodiment, a sirtuin activator is acompound of formula 5 and the attendant definitions, wherein R₂ and R′₃are OH. In a further embodiment, a sirtuin activator is a compound offormula 5 and the attendant definitions, wherein R₂, R₄, and R′₃ are OH.

In a further embodiment, a sirtuin activator is a compound of formula 5and the attendant definitions, wherein Y is CH; Z is O; M is O; R₂ andR′₃ are OH; and R₁, R₃, R₄, R′₁, R′₂, R′₄, and R′₅ are H. In a furtherembodiment, a sirtuin activator is a compound of formula 5 and theattendant definitions, wherein Y is CH; Z is O; M is O; R₂, R₄, and R′₃are OH; and R₁, R₃, R′₁, R′₂, R′₄, and R′₅ are H.

In another embodiment, a sirtuin activator is an anthocyanidin compoundof formula 6:

wherein, independently for each occurrence,

R₃, R₄, R₅, R₆, R₇, R₈, R′₂, R′₃, R′₄, R′₅, and R′₆ represent H, alkyl,aryl, heteroaryl, aralkyl, alkaryl, heteroaralkyl, halide, NO₂, SR, OR,N(R)₂, or carboxyl;

R represents H, alkyl, aryl, heteroaryl, or aralkyl; and

A⁻ represents an anion selected from the following: Cl⁻, Br⁻, or I⁻.

In a further embodiment, a sirtuin activator is a compound of formula 6and the attendant definitions, wherein A⁻ is Cl⁻. In a furtherembodiment, a sirtuin activator is a compound of formula 6 and theattendant definitions, wherein R₃, R₅, R₇, and R′₄ are OH. In a furtherembodiment, a sirtuin activator is a compound of formula 6 and theattendant definitions, wherein R₃, R₅, R₇, R′₃, and R′₄ are OH. In afurther embodiment, a sirtuin activator is a compound of formula 6 andthe attendant definitions, wherein R₃, R₅, R₇, R′₃, R′₄, and R′₅ are OH.

In a further embodiment, a sirtuin activator is a compound of formula 6and the attendant definitions, wherein A⁻ is Cl⁻; R₃, R₅, R₇, and R′₄are OH; and R₄, R₆, R₈, R′₂, R′₃, R′₅, and R′₆ are H. In a furtherembodiment, a sirtuin activator is a compound of formula 6 and theattendant definitions, wherein A⁻ is Cl⁻; R₃, R₅, R₇, R′₃, and R′₄ areOH; and R₄, R₆, R₈, R′₂, R′₅, and R′₆ are H. In a further embodiment, asirtuin activator is a compound of formula 6 and the attendantdefinitions, wherein A^(− is Cl-; R) ₃, R₅, R₇, R′₃, R′₄, and R′₅ areOH; and R₄, R₆, R₈, R′₂, and R′₆ are H.

In a further embodiment, a sirtuin activator is a stilbene, chalcone, orflavone compound represented by formula 7:

wherein, independently for each occurrence,

M is absent or O;

R₁, R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, and R′₅ represent H, alkyl,aryl, heteroaryl, aralkyl, alkaryl, heteroaralkyl, halide, NO₂, SR, OR,N(R)₂, or carboxyl;

R_(a) represents H or the two instances of R_(a) form a bond;

R represents H, alkyl, aryl, heteroaryl, aralkyl; and

n is 0 or 1.

In a further embodiment, a sirtuin activator is an activating compoundrepresented by formula 7 and the attendant definitions, wherein n is 0.In a further embodiment, a sirtuin activator is an activating compoundrepresented by formula 7 and the attendant definitions, wherein n is 1.In a further embodiment, a sirtuin activator is an activating compoundrepresented by formula 7 and the attendant definitions, wherein M isabsent. In a further embodiment, a sirtuin activator is an activatingcompound represented by formula 7 and the attendant definitions, whereinM is O. In a further embodiment, a sirtuin activator is an activatingcompound represented by formula 7 and the attendant definitions, whereinR_(a) is H. In a further embodiment, a sirtuin activator is anactivating compound represented by formula 7 and the attendantdefinitions, wherein M is O and the two R_(a) form a bond.

In a further embodiment, a sirtuin activator is an activating compoundrepresented by formula 7 and the attendant definitions, wherein R₅ is H.In a further embodiment, a sirtuin activator is an activating compoundrepresented by formula 7 and the attendant definitions, wherein R₅ isOH. In a further embodiment, a sirtuin activator is an activatingcompound represented by formula 7 and the attendant definitions, whereinR₁, R₃, and R′₃ are OH. In a further embodiment, a sirtuin activator isan activating compound represented by formula 7 and the attendantdefinitions, wherein R₂, R₄, R′₂, and R′₃ are OH. In a furtherembodiment, a sirtuin activator is an activating compound represented byformula 7 and the attendant definitions, wherein R₂, R′₂, and R′₃ areOH. In a further embodiment, a sirtuin activator is an activatingcompound represented by formula 7 and the attendant definitions, whereinR₂ and R₄ are OH.

In a further embodiment, a sirtuin activator is a compound representedby formula 7 and the attendant definitions, wherein n is 0; M is absent;R_(a) is H; R₅ is H; R₁, R₃, and R′₃ are OH; and R₂, R₄, R′₁, R′₂, R′₄,and R′₅ are H. In a further embodiment, a sirtuin activator is anactivating compound represented by formula 7 and the attendantdefinitions, wherein n is 1; M is absent; R_(a) is H; R₅ is H; R₂, R₄,R′₂, and R′₃ are OH; and R₁, R₃, R′₁, R′₄, and R′₅ are H. In a furtherembodiment, a sirtuin activator is an activating compound represented byformula 7 and the attendant definitions, wherein n is 1; M is O; the twoR_(a) form a bond; R₅ is OH; R₂, R′₂, and R′₃ are OH; and R₁, R₃, R₄,R′₁, R′₄, and R′₅ are H.

Other sirtuin activators include compounds having a formula selectedfrom the group consisting of formulas 8-25 and 30 set forth below.

wherein, independently for each occurrence,

R═H, alkyl, aryl, heterocyclyl, heteroaryl, or aralkyl; and

R′═H, halogen, NO₂, SR, OR, NR₂, alkyl, aryl, or carboxy.

wherein, independently for each occurrence,

R═H, alkyl, aryl, heterocyclyl, heteroaryl, or aralkyl.

wherein, independently for each occurrence,

R′═H, halogen, NO₂, SR, OR, NR₂, alkyl, aryl, aralkyl, or carboxy; and

R═H, alkyl, aryl, heterocyclyl, heteroaryl, or aralkyl.

wherein, independently for each occurrence,

L represents CR₂, O, NR, or S;

R represents H, alkyl, aryl, aralkyl, or heteroaralkyl; and

R′ represents H, halogen, NO₂, SR, OR, NR₂, alkyl, aryl, aralkyl, orcarboxy.

wherein, independently for each occurrence,

L represents CR₂, O, NR, or S;

W represents CR or N;

R represents H, alkyl, aryl, aralkyl, or heteroaralkyl;

Ar represents a fused aryl or heteroaryl ring; and

R′ represents H, halogen, NO₂, SR, OR, NR₂, alkyl, aryl, aralkyl, orcarboxy.

wherein, independently for each occurrence,

L represents CR₂, O, NR, or S;

R represents H, alkyl, aryl, aralkyl, or heteroaralkyl; and

R′ represents H, halogen, NO₂, SR, OR, NR₂, alkyl, aryl, aralkyl, orcarboxy.

wherein, independently for each occurrence,

L represents CR₂, O, NR, or S;

R represents H, alkyl, aryl, aralkyl, or heteroaralkyl; and

R′ represents H, halogen, NO₂, SR, OR, NR₂, alkyl, aryl, aralkyl, orcarboxy.

In a further embodiment, a sirtuin activator is a stilbene, chalcone, orflavone compound represented by formula 30:

wherein, independently for each occurrence,

D is a phenyl or cyclohexyl group;

R₁, R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, and R′₅ represent H, alkyl,aryl, heteroaryl, alkaryl, heteroaralkyl, halide, NO₂, SR, OR, N(R)₂,carboxyl, azide, ether; or any two adjacent R or R′ groups takentogether form a fused benzene or cyclohexyl group;

R represents H, alkyl, aryl, or aralkyl; and

A-B represents an ethylene, ethenylene, or imine group;

provided that when A-B is ethenylene, D is phenyl, and R′₃ is H: R₃ isnot OH when R₁, R₂, R₄, and R₅ are H; and R₂ and R₄ are not OMe when R₁,R₃, and R₅ are H; and R₃ is not OMe when R₁, R₂, R₄, and R₅ are H.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein D is a phenylgroup.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is anethenylene or imine group.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is anethenylene group.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein R₂ is OH.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein R₄ is OH.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein R₂ and R₄ are OH.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein D is a phenylgroup; and A-B is an ethenylene group.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein D is a phenylgroup; A-B is an ethenylene group; and R₂ and R₄ are OH.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; and R′₃ is Cl.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; and R′₃ is OH.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; and R′₃ is H.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; and R′₃ is CH₂CH₃.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; and R′₃ is F.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; and R′₃ is Me.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; and R′₃ is an azide.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; and R′₃ is SMe.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; and R′₃ is NO₂.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; and R′₃ is CH(CH₃)₂.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; and R′₃ is OMe.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; R′₂ is OH; and R′₃ is OMe.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ is OH; R₄ is carboxyl; and R′₃ is OH.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; and R′₃ is carboxyl.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; and R′₃ and R′₄ taken togetherform a fused benzene ring.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; and R₄ is OH.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OCH₂OCH₃; and R′₃ is SMe.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; and R′₃ is carboxyl.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a cyclohexyl ring; and R₂ and R₄ are OH.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; and R₃ and R₄ are OMe.

In a further embodiment, a sirtuin activator is a compound representedby formula 30 and the attendant definitions, wherein A-B is ethenylene;D is a phenyl ring; R₂ and R₄ are OH; and R′₃ is OH.

In one embodiment, sirtuin-modulating compounds of the invention arerepresented by Formula 31:

or a salt thereof, where:

Ring A is optionally substituted; and

Ring B is substituted with at least one carboxy or polycyclic arylgroup.

In another embodiment, sirtuin-modulating compounds of the invention arerepresented by Formula 32:

or a salt thereof, where:

Ring A is optionally substituted;

R₁, R₂, R₃ and R₄ are independently selected from the group consistingof —H, halogen, —OR₅, —CN, —CO₂R₅, —OCOR₅, —OCO₂R₅, —C(O)NR₅R₆,—OC(O)NR₅R₆, —C(O)R₅, —COR₅, —SR₅, —OSO₃H, —S(O)_(n)R₅, —S(O)_(n)OR₅,—S(O)_(n)NR₅R₆, —NR₅R₆, —NR₅C(O)OR₆, —NR₅C(O)R₆ and —NO₂;

R₅ and R₆ are independently —H, a substituted or unsubstituted alkylgroup, a substituted or unsubstituted aryl group or a substituted orunsubstituted heterocyclic group; and

n is 1 or 2.

In certain embodiments, R₁, R₂, R₃ and R₄ are independently selectedfrom the group consisting of —H, —OR₅ and —SR₅, particularly —H and —OR₅(e.g., —H, —OH, —OCH₃).

Ring A is preferably substituted. Suitable substituents include halogens(e.g., bromine), acyloxy groups (e.g., acetoxy), aminocarbonyl groups(e.g., arylaminocarbonyl such as substituted, particularlycarboxy-substituted, phenylaminocarbonyl groups) and alkoxy (e.g.,methoxy, ethoxy) groups.

In yet another aspect, the invention provides novel sirtuin-modulatingcompounds of Formula (III):

or a salt thereof, where:

Ring A is optionally substituted;

R₅ and R₆ are independently —H, a substituted or unsubstituted alkylgroup, a substituted or unsubstituted aryl group or a substituted orunsubstituted heterocyclic group;

R₇, R₉, R₁₀ and R₁₁ are independently selected from the group consistingof —H, halogen, —R₅, —OR₅, —CN, —CO₂R₅, —OCOR₅, —OCO₂R₅, —C(O)NR₅R₆,—OC(O)NR₅R₆, —C(O)R₅, —COR₅, —SR₅, —OSO₃H, —S(O)_(n)R₅, —S(O)_(n)OR₅,—S(O)_(n)NR₅R₆, —NR₅R₆, —NR₅C(O)OR₆, —NR₅C(O)R₆ and —NO₂;

R₈ is a polycyclic aryl group; and

n is 1 or 2.

In certain embodiments, one or more of R₇, R₉, R₁₀ and R₁₁ are —H. Inparticular embodiments, R₇, R₉, R₁₀ and R₁₁ are each —H.

In certain embodiments, R₈ is a heteroaryl group, such as anoxazolo[4,5-b]pyridyl group. In particular embodiments, R₈ is aheteroaryl group and one or more of R₇, R₉, R₁₀ and R₁₁ are —H.

Ring A is preferably substituted. Suitable substituents include halogens(e.g., bromine), acyloxy groups (e.g., acetoxy), aminocarbonyl groups(e.g., arylaminocarbonyl, such as substituted, particularlycarboxy-substituted, phenylaminocarbonyl groups) and alkoxy (e.g.,methoxy, ethoxy) groups, particularly alkoxy groups. In certainembodiments, Ring A is substituted with at least one alkoxy or halogroup, particularly methoxy.

In certain embodiments, Ring A is optionally substituted with up to 3substituents independently selected from (C₁-C₃ straight or branchedalkyl), O—(C₁-C₃ straight or branched alkyl), N(C₁-C₃ straight orbranched allyl)₂, halo, or a 5 to 6-membered heterocycle.

In certain embodiments, Ring A is not substituted with a nitrile orpyrrolidyl group.

In certain embodiments, R₈ is a substituted or unsubstituted bicyclicheteroaryl group, such as a bicyclic heteroaryl group that includes aring N atom and 1 to 2 additional ring heteroatoms independentlyselected from N, O or S. Preferably, R₈ is attached to the remainder ofthe compound by a carbon-carbon bond. In certain such embodiments, 2additional ring heteroatoms are present, and typically at least one ofsaid additional ring heteroatoms is O or S. In certain such embodiments,2 total ring nitrogen atoms are present (with zero or one O or Spresent), and the nitrogen atoms are typically each in a different ring.In certain such embodiments, R₈ is not substituted with acarbonyl-containing moiety, particularly when R₈ is thienopyrimidyl orthienopyridinyl.

In certain such embodiments, R₈ is selected from oxazolopyridyl,benzothienyl, benzofuranyl, indolyl, quinoxalinyl, benzothiazolyl,benzooxazolyl, benzimidazolyl, quinolinyl, isoquinolinyl or isoindolyl.In certain such embodiments, R₈ is selected from thiazolopyridyl,imidazothiazolyl, benzooxazinonyl, or imidazopyridyl.

Particular examples of R₈, where

indicates attachment to the remainder of Formula 33, include:

where up to 2 ring carbons not immediately adjacent to the indicatedattachment point are independently substituted with O—C₁-C₃ straight orbranched alkyl, C₁-C₃ straight or branched alkyl or halo, particularlyC₁-C₃ straight or branched alkyl or halo. In certain embodiments, R₈ is

In certain embodiments, R₈ is

and Ring A is optionally substituted with up to 3 substituentsindependently selected from (C₁-C₃ straight or branched alkyl), O—(C₁-C₃straight or branched allyl), N(C₁-C₃ straight or branched alkyl)₂, halo,or a 5 to 6-membered heterocycle. In certain such embodiments, Ring A isnot simultaneously substituted at the 2- and 6-positions with O—(C₁-C₃straight or branched alkyl). In certain such embodiments, Ring A is notsimultaneously substituted at the 2-, 4- and 6-positions with O—(C₁-C₃straight or branched alkyl). In certain such embodiments, Ring A is notsimultaneously substituted at the 2-, 3-, and 4-positions with O—(C₁-C₃straight or branched alkyl). In certain such embodiments, Ring A is notsubstituted at the 4-position with a 5 to 6-membered heterocycle. Incertain such embodiments, Ring A is not singly substituted at the 3- or4-position (typically 4-position) with O—(C₁-C₃ straight or branchedalkyl). In certain such embodiments, Ring A is not substituted at the4-position with O—(C₁-C₃ straight or branched alkyl) and at the 2- or3-position with C₁-C₃ straight or branched alkyl.

In certain embodiments, R₈ is

and Ring A is optionally substituted with up to 3 substituentsindependently selected from (C₁-C₃ straight or branched alkyl), (C₁-C₃straight or branched haloalkyl, where a haloalkyl group is an alkylgroup substituted with one or more halogen atoms), O—(C₁-C₃ straight orbranched alkyl), N(C₁-C₃ straight or branched alkyl)₂, halo, or a 5 to6-membered heterocycle. In certain such embodiments, Ring A is notsingly substituted at the 3- or 4-position with O—(C₁-C₃ straight orbranched alkyl). In certain such embodiments, Ring A is not substitutedat the 4-position with O—(C₁-C₃ straight or branched allyl) and at the2- or 3-position with C₁-C₃ straight or branched allyl.

In certain embodiments, R₈ is

(e.g., where one or both halo is chlorine) and Ring A is optionallysubstituted with up to 3 substituents independently selected from (C₁-C₃straight or branched alkyl), O—(C₁-C₃ straight or branched alkyl),N(C₁-C₃ straight or branched alkyl)₂, halo, or a 5 to 6-memberedheterocycle, but not singly substituted at the 3-position with O—(C₁-C₃straight or branched alkyl).

In certain embodiments, such as when R₈ has one of the values describedabove, Ring A is substituted with up to 3 substituents independentlyselected from chloro, methyl, O-methyl, N(CH₃)₂ or morpholino. Incertain such embodiments, R₈ is selected from

where up to 2 ring carbons not immediately adjacent to the indicatedattachment point are independently substituted with C₁-C₃ straight orbranched alkyl or halo; each of R₇, R₉, and R₁₁ is —H; and R₁₀ isselected from —H, —CH₂OH, —CO₂H, —CO₂CH₃, —CH₂-piperazinyl, CH₂N(CH₃)₂,—C(O)—NH—(CH₂)₂—N(CH₃)₂, or —C(O)-piperazinyl. In certain suchembodiments, when R₈ is

and Ring A is 3-dimethylaminophenyl, none of R₇, R₉, R₁₀ and R₁₁ is—CH₂—N(CH₃)₂ or —C(O)—NH—(CH₂)₂—N(CH₃)₂, and/or when R₈ is

and Ring A is 3,4 dimethoxyphenyl, none of R₇, R₉, R₁₀ and R₁₁ isC(O)OCH₃ or C(O)OH.

In certain embodiments, such as when R₈ has one of the values describedabove and/or Ring A is optionally substituted as described above, atleast one of R₇, R₉, R₁₀ and R₁₁ is —H. In certain such embodiments,each of R₇, R₉, R₁₀ and R₁₁ is —H.

In certain embodiments, R₇, R₉, R₁₀ or R₁₁ is selected from —C(O)OH,—N(CH₃)₂, —CH₂OH, —CH₂OCH₃, —CH₂-piperazinyl, —CH₂-methylpiperazinyl,—CH₂-pyrrolidyl, —CH₂-piperidyl, —CH₂-morpholino, —CH₂—N(CH₃)₂,—C(O)—NH—(CH₂)_(n)-piperazinyl, C(O)—NH—(CH₂)_(n)-methylpiperazinyl,—C(O)—NH—(CH₂)_(n)-pyrrolidyl, —C(O)—NH—(CH₂)_(n)-morpholino,—C(O)—NH—(CH₂)_(n)-piperidyl, or —C(O)—NH—(CH₂)_(n)—N(CH₃)₂, wherein nis 1 or 2. In certain such embodiments, R₁₀ is selected from —C(O)OH,—N(CH₃)₂, —CH₂OH, —CH₂OCH₃, —CH₂-piperazinyl, —CH₂-methylpiperazinyl,—CH₂-pyrrolidyl, —CH₂-piperidyl, —CH₂-morpholino, —CH₂—N(CH₃)₂,—C(O)—NH—(CH₂)_(n)-piperazinyl,—C(O)—NH—(CH₂)_(n)-methylpiperazinyl-C(O)—NH—(CH₂)_(n)-pyrrolidyl,—C(O)—NH—(CH₂)_(n)-morpholino, —C(O)—NH—(CH₂)_(n)-piperidyl, or—C(O)—NH—(CH₂)_(n)—N(CH₃)₂, wherein n is 1 or 2, and each of R₇, R₉, andR₁₁ is H.

In certain embodiments, Ring A is substituted with a nitrile group or issubstituted at the para position with a 5- or 6-membered heterocycle.Typical examples of the heterocycle include pyrrolidyl, piperidinyl andmorpholinyl.

In one embodiment, sirtuin-modulating compounds of the invention arerepresented by Formula 34:

or a salt thereof, wherein:

two of X¹ to X⁴ are selected from —CR*— and —N—;

the other two of X¹ to X⁴ are —CR*—;

R¹ is a solubilizing group;

R² is a phenyl group optionally substituted with a lower alkyl, loweralkoxy, halogen, nitrile or —CF₃, or R² is a 5- to 6-memberedheterocycle containing an N heteroatom and, optionally, a secondheteroatom selected from N, O or S, wherein said heterocycle isoptionally substituted with methyl or a halogen;

R* is independently selected at each occurrence from —H, lower alkyl orhalogen;

R is —H or —CH₃;

R′ is —CH₃ or a halogen; and

n is an integer from 0-4.

Typically, R is —H and n is 0, such that compounds of Formula 34 arerepresented by Formula 35:

or a salt thereof.

Preferred values in compounds of Formula 34 and 35 are as follows:

two of X¹ to X⁴ are selected from —CR*— and —N—;

the other two of X¹ to X⁴ are —CR*—;

R* is independently selected at each occurrence from —H, lower alkyl orhalogen;

R¹ is a solubilizing group; and

R² is selected from phenyl optionally substituted with one or moresubstituents independently selected from —CN, —F, —Cl and —CF₃, and wheneach of X¹ to X⁴ is —CR*—, R² is additionally selected from a 5- to6-membered heterocycle containing an N heteroatom and, optionally, asecond heteroatom selected from N, O or S, wherein said heterocycle isoptionally substituted with methyl.

In certain embodiments, each of X¹ to X⁴ is —CR*—. In other embodiments,one of X¹ to X⁴ is —N— and the remainder are —CR*—. In certainembodiments, two of X¹ to X⁴ are —N— and the remainder are —CR*—. Incertain embodiments, wherein two of X¹ to X⁴ are —N—, X¹ and X² are —N—.In certain embodiments, wherein two of X¹ to X⁴ are —N—, X¹ and X⁴ are—N—. In certain embodiments, when one of X¹ to X⁴ is —N—, X¹ is —N—. Incertain embodiments, R* is H.

In certain embodiments, such as when each of X¹ to X⁴ is —CR*—, R² isselected from phenyl, fluorophenyl, difluorophenyl, chlorophenyl,methylthiazolyl, pyrimidinyl, pyridyl and pyrazolyl. In certain suchembodiments, R² is selected from phenyl, fluorophenyl, difluorophenyl,chlorophenyl, 2-methylthiazol-4-yl, pyridyl and pyrazol-1-yl.Preferably, R² is phenyl or pyridyl.

In certain embodiments, R¹ is —CH₂—R³ and R³ is a nitrogen-containingheterocycle optionally substituted with one or more substituentsselected from C₁-C₄ alkyl, amino, halogen, methoxy andmethoxy-C₁-C₄-alkyl. In these embodiments, X¹ to X⁴ and R² can have anyof the values described above. In certain such embodiments, R² isphenyl, pyridyl or 3-fluorophenyl; X² and X³ are —CR*— and X¹ and X⁴ areindependently selected from —CR*— or —N—; or both.

In certain embodiments, R¹ is —CH₂—R³; and R³ is selected frompiperazin-1-yl, 4-(methoxyethyl-piperazin-1-yl,3,5-dimethylpiperazin-1-yl, morpholin-4-yl, piperidin-1-yl,4-aminopiperidin-1-yl, pyrrolidin-1-yl, 3-fluoropyrrolidin-1-yl,—NH-(pyrrolidin-3-yl), and 1,4-diaza-bicyclo[2.2.1]heptan-1-yl. In theseembodiments, X¹ to X⁴ and R² can have any of the values described above,but typically R² is phenyl, pyridyl or 3-fluorophenyl; X² and X³ are—CH— and X¹ and X⁴ are independently selected from —CH— or —N—; or both.

In certain such embodiments, R³ is selected from4-(methoxyethyl)-piperazin-1-yl, morpholin-4-yl, piperidin-1-yl and4-aminopiperidin-1-yl. When R³ has these values, R² is typically phenyl,3-fluorophenyl or pyridyl. Also, typically X² and X³ are —CH— and X¹ andX⁴ are independently selected from —CH— or —N—. In particularembodiments, X¹ and X⁴ are independently selected from —CH— or —N—; X²and X³ are —CH—; R² is phenyl, 3-fluorophenyl or pyridyl; and R¹ is—CH₂—R³ where R³ is selected from 4-(methoxyethyl)-piperazin-1-yl,morpholin-4-yl, piperidin-1-yl and 4-aminopiperidin-1-yl.

In certain embodiment, sirtuin-modulating compounds encompassed byFormula 34 are represented by Formula 36:

or a salt thereof, wherein:

one of X¹ to X³ is selected from —CH— and —N—;

the other two of X¹ to X³ are —CH—;

R¹ is a solubilizing group;

R² is a phenyl group optionally substituted with a methyl, halogen or—CF₃, or R² is a 5- to 6-membered heterocycle containing an N heteroatomand, optionally, a second heteroatom selected from N, O or S, whereinsaid heterocycle is optionally substituted with methyl or a halogen;

R is —H or —CH₃;

R′ is —CH₃ or a halogen; and

n is an integer from 0-4.

Typically, R is —H and n is 0, such that compounds of Formula 36 arerepresented by Formula 37:

Preferred values in compounds of Formula 36 and 37 are as follows:

one of X¹ to X³ is selected from —CH— and —N—;

the other two of X¹ to X³ are —CH—;

R¹ is a solubilizing group; and

R² is selected from phenyl and fluorophenyl, and, when each of X¹ to X³is —CH—, R² is additionally selected from a 5- to 6-membered heterocyclecontaining an N heteroatom and, optionally, a second heteroatom selectedfrom N, O or S, wherein said heterocycle is optionally substituted withmethyl.

In certain embodiments, each of X¹ to X³ is —CH—. In other embodiments,one of X¹ to X³ is —N— and the remainder are —CH—. Typically, when oneof X¹ to X³ is —N—, X¹ is N.

In certain embodiments, such as when each of X¹ to X³ is —CH—, R² isselected from phenyl, fluorophenyl, methylthiazolyl, pyrimidinyl,pyridyl and pyrazolyl. In certain such embodiments, R² is selected fromphenyl, fluorophenyl, 2-methylthiazol-4-yl, pyridyl and pyrazol-1-yl.Preferably, R² is phenyl or pyridyl.

In certain embodiments, R¹ is —CH₂—R³ and R³ is a nitrogen-containingheterocycle optionally substituted with one or more substituentsselected from C₁-C₄ alkyl, amino, halogen, methoxy andmethoxy-C₁-C₄-alkyl. In these embodiments, X¹ to X³ and R² can have anyof the values described above. In certain such embodiments, R² isphenyl, pyridyl or 3-fluorophenyl; X² and X³ are —CH— and X¹ is —CH— or—N—; or both.

In certain embodiments, R¹ is —CH₂—R³; and R³ is selected frompiperazin-1-yl, 4-(methoxyethyl-piperazin-1-yl,3,5-dimethylpiperazin-1-yl, morpholin-4-yl, piperidin-1-yl,4-aminopiperidin-1-yl, pyrrolidin-1-yl, 3-fluoropyrrolidin-1-yl,—NH-(pyrrolidin-3-yl), and 1,4-diaza-bicyclo[2.2.1]heptan-1-yl. In theseembodiments, X¹ to X³ and R² can have any of the values described above,but typically R² is phenyl, pyridyl or 3-fluorophenyl; X² and X³ are—CH— and X¹ is —CH— or —N—; or both.

In certain such embodiments, R³ is selected from4-(methoxyethyl)-piperazin-1-yl, morpholin-4-yl, piperidin-1-yl and4-aminopiperidin-1-yl. When R³ has these values, R² is typically phenyl,3-fluorophenyl or pyridyl. Also, typically X² and X³ are —CH— and X¹ is—CH— or —N—. In particular embodiments, X¹ is —CH— or —N—; X² and X³ are—CH—; R² is phenyl, 3-fluorophenyl or pyridyl; and R¹ is —CH₂—R³ whereR³ is selected from 4-(methoxyethyl)-piperazin-1-yl, morpholin-4-yl,piperidin-1-yl and 4-aminopiperidin-1-yl.

In another embodiment, sirtuin-modulating compounds of the invention arerepresented by Formula 38:

or a salt thereof, wherein:

ring A is selected from:

R¹ is a solubilizing group; and

R^(#) is a —H or —O—CH₃.

In yet another embodiment, sirtuin-modulating compounds of the inventionare represented by Formula 39:

or a salt thereof, wherein:

ring B is selected from:

and

R¹ is a solubilizing group.

In some embodiments, a sirtuin pathway activating compound is anycompound described in U.S. Pat. Nos. 7,829,556, 7,855,289, 7,893,086,8,044,198, 8,088,928, and 8,093,401, which are each incorporated hereinby reference in their entirety.

In some embodiments, a sirtuin activating compound is represented by

or a salt thereof.

In some embodiments, a sirtuin-pathway activating compound is a compoundof the formula:

or a salt thereof, wherein:

each of X₇, X₈, X₉ and X₁₀ is independently selected from N, CR²⁰, orCR₁′, wherein:each R²⁰ is independently selected from H or a solubilizing group;each R₁′ is independently selected from H or optionally substitutedC₁-C₃ straight or branched alkyl, wherein when R₁′ is substituted, R₁′is substituted with one or more of —OH, halogen, —OR^(a), —O—COR^(a),—COR^(a), —C(O)R^(a), —CN, —NO₂, —COOH, —COOR^(a), —OCO₂R^(a),—C(O)NR^(a)R^(b), —OC(O)NR^(a)R^(b), —SO₃H, —NH₂, —NHR^(a),—N(R^(a)R^(b)), —COOR^(a), —CHO, —CONH₂, —CONHR^(a), —CON(R^(a)R^(b)),—NHCOR^(a), —NRCOR^(a), —NHCONH₂, —NHCONR^(a)H, —NHCON(R^(a)R^(b)),—NR^(c)CONH₂, —NR^(c)CONR^(a)H, —NR^(c)CON(R^(a)R^(b)), —C(═NH)—NH₂,—C(═NH)—NHR^(a), —C(═NH)—N(R^(a)R^(b)), —C(═NR^(c))—NH₂,—C(═NR^(c))—NHR^(a), —C(═NR^(c))—N(R^(a)R^(b)), —NH—C(═NH)—NH₂,—NH—C(═NH)—NHR^(a), —NH—C(═NH)—N(R^(a)R^(b)), —NH—C(═NR^(c))—NH₂,—NH—C(═NR^(c))—NHR^(a), —NH—C(═NR^(c))—N(R^(a)R^(b)),—NR^(d)H—C(═NH)—NH₂, —NR^(d)—C(═NH)—NHR^(a),—NR^(d)—C(═NH)—N(R^(a)R^(b)), —NR^(d)—C(═NR^(c))—NH₂,—NR^(d)—C(═NR^(c))—NHR^(a), —NR^(d)—C(═NR^(c))—N(R^(a)R^(b)), —NHNH₂,—NHNHR^(a), —NHR^(a)R^(b), —SO₂NH₂—SO₂NHR_(a), —SO₂NR^(a)R^(b),—CH═CHR^(a), —CH═CR^(a)R^(b), —CR^(c)═CR^(a)R^(b), CR^(c)═CHR^(a),—CR^(c)═CR^(a)R^(b), —CCR^(a), —SH, —SO_(k)R^(a), —S(O)_(k)OR^(a) and—NH—C(═NH)—NH₂, whereink is 0, 1 or 2;R^(a)-R^(d) are each independently an aliphatic, substituted aliphatic,benzyl, substituted benzyl, aromatic or substituted aromatic group; and—NR^(a)R^(b), taken together, can also form a substituted orunsubstituted non-aromatic heterocyclic group;wherein a non-aromatic heterocyclic group, benzylic group or aryl groupcan also have an aliphatic or substituted aliphatic group as asubstituent; a substituted aliphatic group can also have a non-aromaticheterocyclic ring, a substituted non-aromatic heterocyclic ring, benzyl,substituted benzyl, aryl or substituted aryl group as a substituent; anda substituted aliphatic, non-aromatic heterocyclic group, substitutedaryl, or substituted benzyl group can have more than one substituent;one of X₇, X₈, X₉ and X₁₀ is N and the others are selected from CR²⁰ orCR₁′; and zero to one R²⁰ is a solubilizing group;R¹⁹ is selected from:

wherein:each Z₁₀, Z₁₁, Z₁₂ and Z₁₃ is independently selected from N, CR²⁰, orCR₁′; andeach Z₁₄, Z₁₅ and Z₁₆ is independently selected from N, NR₁′, S, O,CR²⁰, or CR₁′, wherein:zero to two of Z₁₀, Z₁₁, Z₁₂ and Z₁₃ are N;at least one of Z₁₄, Z₁₅ and Z₁₆ is N, NR₁′, S or O;zero to one of Z₁₄, Z₁₅ and Z₁₆ is S or O;zero to two of Z₁₄, Z₁₅ and Z₁₆ are N or NR₁′;zero to one R²⁰ is a solubilizing group;zero to one R₁′ is an optionally substituted C₁-C₃ straight or branchedalkyl; andR²¹ is selected from —NR₁′—C(O)—, —NR₁′—S(O)₂—, —NR₁′—C(O)—NR₁′—,—NR₁′—C(S)—NR₁′—, —NR₁′—C(S)—NR₁′—CR₁′R₁′—, —NR₁′—C(O)—CR₁′R₁′—NR₁′—,—NR₁′—C(═NR₁′)—NR₁′—, —C(O)—NR₁′—, —C(O)—NR₁′—S(O)₂—, —NR₁′—, —CR₁′R₁′—,—NR₁′—C(O)—CR₁′═CR₁′—, —NR₁′—S(O)₂—NR₁′—, —NR₁′—C(O)—NR₁′—S(O)₂—,—NR₁′—CR₁′R₁′—C(O)—NR₁′—, —CR₁′R₁′—C(O)—NR₁′—,—NR₁′—C(O)—CR₁′═CR₁′—CR₁′R₁′—, —NR₁′—C(═N—CN)—NR₁′—,—NR₁′—C(O)—CR₁′R₁′—O—, —NR₁′—C(O)—CR₁′R₁′—CR₁′R₁′—O—,—NR₁′—S(O)₂—CR₁′R₁′—, —NR₁′—S(O)₂—CR₁′R₁′—CR₁′R₁′—,—NR₁′—C(O)—CR₁′R₁′—CR₁′R₁′—, —NR₁′—C(S)—NR₁′—CR₁′R₁′—CR₁′R₁′—,—NR₁′—C(O)—O— or —NR₁′—C(O)—CR₁′R₁′—; andR³¹ is selected from an optionally substituted monocyclic or bicyclicaryl, or an optionally substituted monocyclic or bicyclic heteroaryl,with the proviso that:when X₇ is N, R¹⁹ is

and each of Z₁₀, Z₁₁, Z₁₂ and Z₁₃ is independently selected from CR²⁰ orCR₁′, then:

a) at least one of X₈, X₉ and X₁₀ is C—(C₁-C₃ straight or branchedalkyl) or C-(solubilizing group); orb) at least one of Z₁₀, Z₁₁, Z₁₂ and Z₁₃ is CR²⁰, wherein R²⁰ is asolubilizing group.

In some embodiments, a sirtuin-pathway activating compound is a compoundof the formula:

or a salt thereof, wherein:

two of X¹ to X⁴ are selected from —CR*— and —N—;the other two of X¹ to X⁴ are —CR*—;R* is independently selected at each occurrence from —H, lower alkyl orhalogen;R¹ is a solubilizing group; andR² is selected from phenyl optionally substituted with one or moresubstituents independently selected from —CN, —F, —Cl and —CF₃, and wheneach of X¹ to X⁴ is —CR*—, R² is additionally selected from a 5- to6-membered heterocycle containing an N heteroatom and, optionally, asecond heteroatom selected from N, O or S, wherein said heterocycle isoptionally substituted with methyl.

In some embodiments, a sirtuin-pathway activating compound is a compoundof the formula:

or a salt thereof.

In some embodiments, a sirtuin-pathway activating compound is a compoundof the formula:

or a salt thereof, wherein:

each of X₇, X₈, X₉ and X₁₀ is independently selected from N, CR²⁰, andCR₁′, wherein:each R²⁰ is independently selected from H and a solubilizing group;each R₁′ is independently selected from H and optionally substitutedC₁-C₃ straight or branched alkyl, wherein when R₁′ is substituted, R₁′is substituted with one or more of —OH, halogen, —OR^(a), —O—COR^(a),—COR^(a), —C(O)R^(a), —CN, —NO₂, —COOH, —COOR^(a), —OCO₂R^(a),—C(O)NR^(a)R^(b), —OC(O)NR^(a)R^(b), —SO₃H, —NH₂, —NHR^(a),—N(R^(a)R^(b)), —COOR^(a), —CHO, —CONH₂, —CONHR^(a), —CON(R^(a)R^(b)),—NHCOR^(a), —NRCOR^(a), —NHCONH₂, —NHCONR^(a)H, —NHCON(R^(a)R^(b)),—NR^(c)CONH₂, —NR^(c)CONR^(a)H, —NR^(c)CON(R^(a)R^(b)), —C(═NH)—NH₂,—C(═NH)—NHR^(a), —C(═NH)—N(R^(a)R^(b)), —C(═NR^(c))—NH₂,—C(═NR^(c))—NHR^(a), —C(═NR^(c))—N(R^(a)R^(b)), —NH—C(═NH)—NHR^(a),—NH—C(═NH)—N(R^(a)R^(b)), —NH—C(═NR^(c))—NH₂, —NH—C(═NR^(c))—NHR^(a),—NH—C(═NR^(c))—N(R^(a)R^(b)), —NR^(d)—C(═NH)—NH₂,—NR^(d)—C(═NH)—NHR^(a), —NR^(d)—C(═NH)—N(R^(a)R^(b)),—NR^(d)—C(═NR^(c))—NH₂, —NR^(d)—C(═NR^(c))—NHR^(a),—NR^(d)—C(═NR^(c))—N(R^(a)R^(b)), —NHNH₂, —NHNHR^(a), —SO₂NH₂,—SO₂NHR^(a), —SO₂NR^(a)R^(b), —CH═CHR^(a), —CH═CR^(a)R_(b),—CR^(c)═CR^(a)R^(b), CR^(c)═CHR^(a), —CR^(c)═CR^(a)R^(b), —CCR^(a), —SH,—SO_(k)R^(a), —S(O)_(k)OR^(a) and —NH—C(═NH)—NH₂, whereink is 0, 1 or 2;R^(a)-R^(d) are each independently an aliphatic, substituted aliphatic,benzyl, substituted benzyl, aromatic or substituted aromatic group; and—NR^(a)R^(b), taken together, can also form a substituted orunsubstituted non-aromatic heterocyclic group;wherein a non-aromatic heterocyclic group, benzylic group or aryl groupcan also have an aliphatic or substituted aliphatic group as asubstituent; a substituted aliphatic group can also have a non-aromaticheterocyclic ring, a substituted non-aromatic heterocyclic ring, benzyl,substituted benzyl, aryl or substituted aryl group as a substituent; anda substituted aliphatic, non-aromatic heterocyclic group, substitutedaryl, or substituted benzyl group can have more than one substituent;one of X₇, X₈, X₉ and X₁₀ is N and the others are selected from CR²⁰ andCR₁′; andzero to one R²⁰ is a solubilizing group;R¹⁹ is selected from:

wherein:each Z₁₀, Z₁₁, Z₁₂ and Z₁₃ is independently selected from CR²⁰ and CR₁′;wherein:zero to one R²⁰ is a solubilizing group;zero to one R₁′ is an optionally substituted C₁-C₃ straight or branchedalkyl; andR²¹ is selected from —NR₁′—C(O)—, and —C(O)—NR₁′ andR³¹ is selected from an optionally substituted monocyclic or bicyclicaryl, and an optionally substituted monocyclic or bicyclic heteroaryl,with the proviso that:said compound is not:

In some embodiments, a sirtuin-pathway activating compound is a compoundof the formula:

or a salt thereof, wherein:

each of R²³ and R²⁴ is independently selected from H, —CH₃ and asolubilizing group;R²⁵ is selected from H and a solubilizing group; and

R¹⁹ is:

wherein:

each Z₁₀, Z₁₁, Z₁₂ and Z₁₃ is independently selected from CR²⁰ and CR₁″;wherein:zero to one R²⁰ is a solubilizing group; andzero to one R₁″ is an optionally substituted C₁-C₃ straight or branchedalkyl;each R²⁰ is independently selected from H and a solubilizing group;R²¹ is selected from —NR₁′—C(O)— and —C(O)—NR₁′; andeach R₁′ is independently selected from H and optionally substitutedC₁-C₃ straight or branched alkyl, wherein when R₁′ and/or R₁″ issubstituted, R₁′ and/or R₁″ is substituted with one or more of —OH,halogen, —OR^(a), —O—COR^(a), —COR^(a), —C(O)R^(a), —CN, —NO₂, —COOH,—COOR^(a), —OCO₂R^(a), —C(O)NR^(a)R^(b), —OC(O)NR^(a)R^(b), —SO₃H, —NH₂,—NHR^(a), —N(R^(a)R^(b)), —COOR^(a), —CHO, —CONH₂, —CONHR^(a),—CON(R^(a)R^(b)), —NHCOR^(a), —NRCOR^(a), —NHCONH₂, —NHCONR^(a)H,—NHCON(R^(a)R^(b)), —NR^(c)CONH₂, —NR^(c)CONR^(a)H,—NR^(c)CON(R^(a)R^(b)), —C(═NH)—NH₂, —C(═NH)—NHR^(a),—C(═NH)—N(R^(a)R^(b)), —C(═NR^(c))—NH₂, —C(═NR^(c))—NHR^(a),—C(═NR^(c))—N(R^(a)R^(b)), —NH—C(═NH)—NHR^(a), —NH—C(═NH)—N(R^(a)R^(b)),—NH—C(═NR^(c))—NH₂, —NH—C(═NR^(c))—NHR^(a),—NH—C(═NR^(c))—N(R^(a)R^(b)), —NR^(d)—C(═NH)—NH₂,—NR^(d)—C(═NH)—NHR^(a), —NR^(d)—C(═NH)—N(R^(a)R^(b)),—NR^(d)—C(═NR^(c))—NH₂, —NR^(d)—C(═NR^(c))—NHR^(a),—NR^(d)—C(═NR^(c))—N(R^(a)R^(b)), —NHNH₂, —NHNHR^(a), —SO₂NH₂,—SO₂NHR_(a), —SO₂NR^(a)R^(b), —CH═CHR^(a), —CH═CR^(a)R^(b),—CR^(c)═CR^(a)R^(b), CR^(c)═CHR^(a), —CR^(c)═CR^(a)R^(b), —CCR^(a), —SH,—SO_(k)R^(a), —S(O)_(k)OR^(a) and —NH—C(═NH)—NH₂, whereink is 0, 1 or 2;R^(a)-R^(d) are each independently an aliphatic, substituted aliphatic,benzyl, substituted benzyl, aromatic or substituted aromatic group; and—NR^(a)R^(b), taken together, can also form a substituted orunsubstituted non-aromatic heterocyclic group;wherein a non-aromatic heterocyclic group, benzylic group or aryl groupcan also have an aliphatic or substituted aliphatic group as asubstituent; a substituted aliphatic group can also have a non-aromaticheterocyclic ring, a substituted non-aromatic heterocyclic ring, benzyl,substituted benzyl, aryl or substituted aryl group as a substituent; anda substituted aliphatic, non-aromatic heterocyclic group, substitutedaryl, or substituted benzyl group can have more than one substituent;andR³¹ is selected from an optionally substituted monocyclic or bicyclicaryl, and an optionally substituted monocyclic or bicyclic heteroaryl,with the proviso that R³¹ is not 2,4-dimethoxyphenyl.

In various other embodiments, compositions are formulated such that theydo not contain (or exclude) one or more of the following ingredients:caffeine, green tea extract or extracts from guarana seed or guaranaplants.

In other embodiments, the sirtuin-pathway activator or AMPK pathwayactivator can be irisin, quinic acid, cinnamic acid, ferulic acid,fucoxanthin, a biguanide (such as metformin), rosiglitazone, or anyanalog thereof. Alternatively the sirtuin-pathway activator or AMPKpathway activator can be isoflavones, pyroloquinoline (PQQ), quercetin,L-carnitine, lipoic acid, coenzyme Q10, pyruvate,5-aminoimidazole-4-carboxamide ribotide (ALCAR), bezfibrate, oltipraz,and/or genistein. In some embodiments, the sirtuin pathway activator isa PDE inhibitor.

In some embodiments, the composition can comprise combinations ofmetformin, resveratrol, and a branched chain amino acid or metabolitethereof. For example, a composition can comprise metformin, resveratrol,and HMB or the composition can comprise metformin, resveratrol, andleucine. Combinations of metformin, resveratrol, and a branched chainamino acid can cause an increase in fatty acid oxidation of over 700,800, 900, 1000, 1200, 1400, 1600, or 1800%.

In some embodiments, the composition can comprise synergisticcombinations of sirtuin pathway activators. For example, a compositioncan comprise synergistic amounts of metformin and a PDE inhibitor. Insome embodiments, the composition comprises metformin and caffeine.

In some embodiments, the sirtuin-pathway activator can be an agent thatstimulates the expression of the Fndc5, PGC1-α, or UCP1. The expressioncan be measured in terms of the gene or protein expression level.Alternatively, the sirtuin pathway activator can be irisin. Methods forincreasing the level of irisin are described in Boström et al., “APGC1-α-dependent myokine that drives brown-fat-like development of whitefat and thermogenesis,” Nature, Jan. 11, 2012.

In some embodiments, the activator is a flavones or chalcone. In oneembodiment, exemplary sirtuin activators are those described in Howitzet al. (2003) Nature 425: 191 and include, for example, resveratrol(3,5,4′-Trihydroxy-trans-stilbene), butein(3,4,2′,4′-Tetrahydroxychalcone), piceatannol(3,5,3′,4′-Tetrahydroxy-trans-stilbene), isoliquiritigenin(4,2′,4′-Trihydroxychalcone), fisetin (3,7,3′,4′-Tetrahyddroxyflavone),quercetin (3,5,7,3′,4′-Pentahydroxyflavone), Deoxyrhapontin(3,5-Dihydroxy-4′-methoxystilbene 3-O-β-D-glucoside); trans-Stilbene;Rhapontin (3,3′,5-Trihydroxy-4′-methoxystilbene 3-O-β-D-glucoside);cis-Stilbene; Butein (3,4,2′,4′-Tetrahydroxychalcone);3,4,2′4′6′-Pentahydroxychalcone; Chalcone;7,8,3′,4′-Tetrahydroxyflavone; 3,6,2′,3′-Tetrahydroxyflavone;4′-Hydroxyflavone; 5,4′-Dihydroxyflavone 5,7-Dihydroxyflavone; Morin(3,5,7,2′,4′-Pentahydroxyflavone); Flavone; 5-Hydroxyflavone;(−)-Epicatechin (Hydroxy Sites: 3,5,7,3′,4′); (−)-Catechin (HydroxySites: 3,5,7,3′,4′); (−)-Gallocatechin (Hydroxy Sites: 3,5,7,3′,4′,5′)(+)-Catechin (Hydroxy Sites: 3,5,7,3′,4′);5,7,3′,4′,5′-pentahydroxyflavone; Luteolin(5,7,3′,4′-Tetrahydroxyflavone); 3,6,3′,4′-Tetrahydroxyflavone;7,3′,4′,5′-Tetrahydroxyflavone; Kaempferol(3,5,7,4′-Tetrahydroxyflavone); 6-Hydroxyapigenin(5,6,7,4′-Tetrahydoxyflavone); Scutellarein); Apigenin(5,7,4′-Trihydroxyflavone); 3,6,2′,4′-Tetrahydroxyflavone;7,4′-Dihydroxyflavone; Daidzein (7,4′-Dihydroxyisoflavone); Genistein(5,7,4′-Trihydroxyflavanone); Naringenin (5,7,4′-Trihydroxyflavanone);3,5,7,3′,4′-Pentahydroxyflavanone; Flavanone; Pelargonidin chloride(3,5,7,4′-Tetrahydroxyflavylium chloride); Hinokitiol (b-Thujaplicin;2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1-one); L-(+)-Ergothioneine((S)-a-Carboxy-2,3-dihydro-N,N,N-trimethyl-2-thioxo-1H-imidazole-4-ethanaminiuminner salt); Caffeic Acid Phenyl Ester; MCI-186(3-Methyl-1-phenyl-2-pyrazolin-5-one); HBED (N,N′-Di-(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid-H2O); Ambroxol(trans-4-(2-Amino-3,5-dibromobenzylamino) cyclohexane-HCl; and U-83836E((−)-2-((4-(2,6-di-1-Pyrrolidinyl-4-pyrimidinyl)-1-piperzainyl)methyl)-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol.2HCl).Analogs and derivatives thereof can also be used.

The subject application provides compositions useful for inducing anincrease in fatty acid oxidation and mitochondrial biogenesis in asubject. Such compositions contain: HMB in combination with resveratrol;leucine in combination with resveratrol; both leucine and HMB incombination with resveratrol; KIC in combination with resveratrol; bothKIC and HMB in combination with resveratrol; both KIC and leucine incombination with resveratrol; or KIC, HMB and leucine in combinationwith resveratrol.

Phosphodiesterase Inhibitors

In some embodiments, the sirtuin pathway activator modulates theactivity of phosphodiesterase (PDE). In some embodiments, the sirtuinpathway activator is a PDE inhibitor, such as a non-specific PDEinhibitor. PDE inhibitors can be naturally occurring or non-naturallyoccurring (e.g. manufactured), and may be provided in the form of anatural source comprising the PDE inhibitor, or an extract thereof (e.g.purified). Examples of non-specific PDE inhibitors include, but are notlimited to, caffeine, theophylline, theobromine,3-isobutyl-1-methylxanthine (IBMX), pentoxifylline(3,7-dihydro-3,7-dimethyl-1-(5oxohexyl)-1H-purine-2,6-dione),aminophylline, paraxanthine, and salts, derivatives, metabolites,catabolites, anabolites, precursors, and analogs thereof. Non-limitingexamples of natural sources of PDE inhibitors include coffee, tea,guarana, yerba mate, cocoa, and chocolate (e.g. dark chocolate).

In some embodiments, a PDE inhibitor is administered in place of or inaddition to resveratrol or other sirtuin pathway activator. In someembodiments, compositions comprising one or more components describedherein comprise a PDE inhibitor in place of or in addition toresveratrol or other sirtuin pathway activator. Typically, a PDEinhibitor is provided in an amount that is synergistic with one or moreother components of a composition or method of treatment.

Branched Chain Amino Acids

The invention provides for compositions that include branched chainamino acids. Branched chain amino acids can have aliphatic side chainswith a branch carbon atom that is bound to two or more other atoms. Theother atoms may be carbon atoms. Examples of branched chain amino acidsinclude leucine, isoleucine, and valine. Branched chain amino acids mayalso include other compounds, such as 4-hydroxyisoleucine. In someembodiments, the compositions may be substantially free of one or more,or all of non-branched chain amino acids. For example, the compositionscan be free of alanine, arginine, asparagine, aspartic acid, cysteine,glutamic acid, glutamine, glycine, histidine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, and/or tyrosine.In some embodiments, the compositions may be substantially free ofisoleucine and/or valine.

Without being limited to theory, ingestion of branched chain aminoacids, such as leucine, can stimulate tissue protein synthesis via bothmTOR-dependent and -independent pathways, as well as to exert anantiproteolytic effect. These effects predominate in muscle, but alsocan manifest in other tissues, including adipose tissue. Given theenergetic cost of protein synthesis and turnover, leucine may increasefatty acid oxidation and net energy utilization and attenuate adiposity.Indeed, leucine has been reported to exert a thermogenic effect and toaugment weight and adipose tissue loss during energy restriction. Also,leucine and leucine-rich diets can favorably modulate inflammatorycytokine patterns in adipocytes and mice.

In some embodiments, any of the compositions described herein caninclude salts, derivatives, metabolites, catabolites, anabolites,precursors, and analogs of any of the branched chain amino acids. Forexample, the metabolites of branched chain amino acids can includehydroxymethylbutyrate (HMB), α-hydroxyisocaproic acid, andketo-isocaproic acid (KIC), keto isovalerate, and keto isocaproate.Non-limiting exemplary anabolites of branched chain amino acids caninclude glutamate, glutamine, threonine, α-ketobytyrate,α-aceto-α-hydroxy butyrate, α,β-dihydroxy-β-methylvalerate,α-keto-β-methylvalerate, α,β-dihydroxy isovalerate, and α-ketoisovalerate.

In certain embodiments of the invention, any of the compositionsdisclosed herein can be formulated such that they do not contain (orexclude) one or more amino acids selected from the group consisting oflysine, glutamate, proline, arginine, valine, isoleucine, aspartic acid,asparagine, glycine, threonine, serine, phenylalanine, tyrosine,histidine, alanine, tryptophan, methionine, glutamine, taurine,carnitine, cystine and cysteine. The compositions can be substantiallyfree of any non-branched chain amino acids. The mass or molar amount ofa non-branched chain amino acid can be less than 0.01, 0.1, 0.5, 1, 2,or 5% of the total composition.

Vitamin B6

Without being limited to any particular theory or mode of action,elevations in the active B6 metabolite (pyridoxal phosphate) can reducethe tone and activity of the adipocyte calcium channel. Intracellularfree Ca2+ is a primary regulator of adipocyte fatty acid synthaseexpression and activity, which can result in a suppression of both theexpression and activity of fatty acid synthase, which in turn is one ofthe rate limiting steps in neutral lipid synthesis in adipocytes.

As used herein, vitamin B6 includes its different forms, includingpyridoxine, pyridoxine 5′-phosphate, pyridoxal, pyridoxal phosphate,pyridoxal 5′-phosphate, pyridoxamine, pyridoxamine 5′-phosphate. Inother embodiments, vitamin B6 can also include 4-pyridoxic acid, whichis a catabolite of the above forms of vitamin B6 that is excreted. Thecompositions described herein can include any one or more of these formsof vitamin B6.

The active form of vitamin B6 in the body is pyridoxal 5-phosphate,which is a coenzyme for all transamination and some decarboxylation anddeamination reactions. Furthermore, pyridoxal 5-phosphate is required asa coenzyme for all transamination reactions which occur in the body(Peterson D L, Martinez-Carrion M. The mechanism of transamination.Function of the histidyl residue at the active site of supernatantaspartate transaminase. J Biol Chem. 1970 Feb. 25; 245(4):806-13).

In some embodiments, any of the compositions described herein caninclude salts, derivatives, metabolites, catabolites, anabolites,precursors, and analogs of any of the forms of vitamin B6. Exemplarycatabolites of vitamin B6 include2-methyl-3-hydroxy-5-formylpyridine-4-carboxylate and3-hydroxy-2-methylpyridine-4,5,-dicarboxylate. Exemplary analogs ofvitamin B6 are described in U.S. Pat. Nos. 7,230,009, and 6,369,042.Exemplary precursors of vitamin B6 are described in U.S. Pat. No.7,495,101.

Pharmaceutically Active Agents

The combination compositions can further include one or morepharmaceutically active agents. Examples of therapeutically activeagents include ibuprofen, aldoril, and gemfebrozil, verapamil, maxzide,diclofenac and metrolol, maproltiline, triazolam and minoxidil. Forexample, the combination compositions can comprise a pharmaceuticallyactive anti-diabetic agent, weight loss agent, or calcium regulationagent. U.S. Pat. No. 7,109,198 and U.S. Patent Application No.20090142336 describe a variety of pharmaceutically active agents ortherapeutically active agents suitable for inclusion in a combinationcomposition described herein. Examples of anti-diabetic agents includebiguanides (such as metformin), thiazoladinediones and meglitinides(such as repaglinide, pioglitazone, and rosiglitazone), alphaglucosidease inhibitors (such as acarbose), sulfonylureas (such astolbutamide, acetohexamide, tolazamide, chlorpropamide, glipizide,glyburide, glimepiride, gliclazide), incretins, ergot alkaloids (such asbromocriptine), and DPP inhibitors (such as sitagliptin, vildagliptin,saxagliptin, lingliptin, dutogliptin, gemigliptin, alogliptin, andberberine). The anti-diabetic agent can be an oral anti-diabetic agent.The anti-diabetic agent can also be injectable anti-diabetic drugs,including insulin, amylin analogues (such as pramlintide), and inretinmimetics (such as exenatide and liraglutide). Examples of anti-obesitytherapeutic agents include lipase inhibitors (such as Orlistat),dopaminergic, noradrenergic, and serotoninergic compounds, cannabinoidreceptor antagonists (such as rimonabant), exenatide, pramlintide, andCNS agents (such as topimerate). These examples are provided fordiscussion purposes only, and are intended to demonstrate the broadscope of applicability of the invention to a wide variety of drugs. Itis not meant to limit the scope of the invention in any way.

In some embodiments, one or more components described herein, such asresveratrol, leucine, HMB, and KIC can be combined with two or morepharmaceutically active agents. For example, a sirtuin pathway activatorcan be combined with glipizide and metformin, glyburide and metformin,pioglitazone and glimepiride, pioglitazone and metformin, repaglinideand metformin, rosiglitazone and glimepiride, rosiglitazone andmetformin, or sitagliptin and metformin.

The amount of pharmaceutical agent, or any other component used in acombination composition described herein, can be a used in an amountthat is sub-therapeutic. In some embodiments, using sub-therapeuticamounts of an agent or component can reduce the side-effects of theagent. Use of sub-therapeutic amounts can still be effective,particularly when used in synergy with other agents or components.

A sub-therapeutic amount of the agent or component can be such that itis an amount below which would be considered therapeutic. For example,FDA guidelines can suggest a specified level of dosing to treat aparticular condition, and a sub-therapeutic amount would be any levelthat is below the FDA suggested dosing level. The sub-therapeutic amountcan be about 1, 5, 10, 15, 20, 25, 30, 35, 50, 75, 90, or 95% less thanthe amount that is considered to be a therapeutic amount. Thetherapeutic amount can be assessed for individual subjects, or forgroups of subjects. The group of subjects can be all potential subjects,or subjects having a particular characteristic such as age, weight,race, gender, or physical activity level.

In the case of metformin hydrochloride, the physician suggested startingdose is 1000 mg daily, with subject specific dosing having a range of500 mg to a maximum of 2500 mg daily (metformin hydrocholrideextended-release tablets labelwww.accessdata.fda.gov/drugsatfda_docs/label/2008/021574s0101bl.pdf).The particular dosing for a subject can be determined by a clinician bytitrating the dose and measuring the therapeutic response. Thetherapeutic dosing level can be determined by measuring fasting plasmaglucose levels and measuring glycosylated hemoglobin. A sub-therapeuticamount can be any level that would be below the recommended dosing ofmetformin. For example, if a subject's therapeutic dosing level isdetermined to be 700 mg daily, a dose of 600 mg would be asub-therapeutic amount. Alternatively, a sub-therapeutic amount can bedetermined relative to a group of subjects rather than an individualsubject. For example, if the average therapeutic amount of metformin forsubjects with weights over 300 lbs is 2000 mg, then a sub-therapeuticamount can be any amount below 2000 mg. In some embodiments, the dosingcan be recommended by a healthcare provider including, but not limitedto a patient's physician, nurse, nutritionist, pharmacist, or otherhealth care professional. A health care professional may include aperson or entity that is associated with the health care system.Examples of health care professionals may include surgeons, dentists,audiologists, speech pathologists, physicians (including generalpractitioners and specialists), physician assistants, nurses, midwives,pharmaconomists/pharmacists, dietitians, therapists, psychologists,physical therapists, phlebotomists, occupational therapists,optometrists, chiropractors, clinical officers, emergency medicaltechnicians, paramedics, medical laboratory technicians, radiographers,medical prosthetic technicians social workers, and a wide variety ofother human resources trained to provide some type of health careservice.

Dosing Amounts

In some embodiments, a composition comprises an amount of a sirtuinpathway activator, such as a a polyphenol (e.g. resveratrol). The amountof sirtuin pathway activator may be a subtheratpeutic amount, and/or anamount that is synergistic with one or more other compounds in thecomposition or one or more othe comounds administered simultaneously orin close temporal proximity with the composition. In some embodiments,the sirtuin pathway activator is administered in a low dose, a mediumdose, or a high dose, which describes the relationship between twodoses, and generally do not define any particular dose range. Forexample, a daily low dose of resveratrol may comprise about, less thanabout, or more than about 0.5 mg/kg, 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5mg/kg, 10 mg/kg, 12.5 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 50 mg/kg, 75mg/kg, 100 mg/kg, or more; a daily medium dose of resveratrol maycomprise about, less than about, or more than about 20 mg/kg, 25 mg/kg,50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200mg/kg, 250 mg/kg, or more; and a daily high dose of resveratrol maycomprise about, less than about, or more than about 150 mg/kg, 175mg/kg, 200 mg/kg, 225 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg,or more.

In some embodiments of the invention, the following amounts of leucine,HMB, KIC, vitamin D, vitamin K2, and/or resveratrol are to beadministered to a subject: leucine about, less than about, or more thanabout 0.5-3.0 g/day (e.g. 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, ormore g/day); HMB about, less than about, or more than about 0.20-3.0g/day (e.g. 0.2, 0.4, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, or more g/day); KICabout, less than about, or more than about 0.2-3.0 g/day (e.g. 0.2, 0.4,0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, or more g/day); vitamin Dabout, less than about, or more than about 2.5-25 μg/day (e.g. 2.5, 5,7.5, 10, 12.5, 15, 17.5, 20, 25, or more μg/day); vitamin K2 about, lessthan about, or more than about 5-200 μg/day (e.g. 5, 10, 25, 50, 75,100, 150, 200, or more μg/day); and/or resveratrol about, less thanabout, or more than about 10-500 mg/day (e.g. 10, 25, 50, 51, 75, 100,150, 200, 250, 300, 350, 400, 450, 500, or more mg/day). Thus, oneembodiment provides a composition comprising leucine in an amount ofabout 0.75 to about 3.0 g (0.75 to 3.0 g) and resveratrol in an amountbetween about 50 and about 500 mg (or 50 to 500 mg). Another embodimentprovides a composition comprising HMB in an amount of 0.40-3.0 g (or0.40 to 3.0 g) and resveratrol in an amount between 50-500 mg (or 50 to500 mg). Another embodiment provides for a composition comprisingleucine in an amount of about 0.75-about 3.0 g (or 0.75 to 3.0 g), HMBin an amount of about 0.40 and about 3.0 g (or 0.40 to 3.0 g) andresveratrol in an amount between about 50 and about 500 mg (or 50 to 500mg). In some embodiments, a composition further comprises a PDEinhibitor in a synergizing amount. In some embodiments, a compositionfurther comprises a sirtuin pathway activator in a synergizing amount.In some embodiments, resveratrol is replaced with a PDE inhibitor orother sirtuin pathway activator in a synergizing amount. In compositionscomprising a PDE inhibitor or methods comprising administration of a PDEinhibitor (separately from or concurrently with one or more othercomponents), the PDE inhibitor may be provided in an amount thatproduces a peak plasma concentration of about, less than about, or morethan about 0.1, 1, 5, 10, 25, 50, 100, 500, 1000, 2500, 5000, 10000, ormore nM.

Another aspect of the invention provides compositions comprisingsynergizing amounts of resveratrol and leucine; resveratrol and HMB;resveratrol, leucine and HMB; resveratrol and KIC; resveratrol, KIC andleucine; resveratrol, KIC, and HMB; or resveratrol, KIC, leucine andHMB. In some embodiments, a synergizing amount of resveratrol is anamount of at least 35 mg of resveratrol and no more than 500 (or about500) mg resveratrol (e.g. 35, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, or 500 mg resveratrol) in combination with leucine and/or HMB.Synergizing amounts of leucine and/or KIC in a composition containingleucine and/or KIC and resveratrol can range from about, less thanabout, or more than about 0.50 to 3.0 g (or about 0.50 to about 3.0 g;e.g. 0.5, 0.75, 1, 1.5, 2, 2.5, 3 or more grams) or 0.75 to 3.0 g (orabout 0.75 to 3.0 g; e.g. 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3 or moregrams). Synergizing amounts of HMB provided in a composition containingHMB and resveratrol contains HMB in an amount of about, less than about,or more than about 0.20-3.0 g (or about 0.20 to about 3.0 g; e.g. 0.2,0.4, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, or more grams). In some embodiments,where combinations of leucine and KIC are used in a composition, thetotal amount of leucine and KIC is less than, or equal to, 3.0 g (orless than about 3.0 g; e.g. less than about 0.7, 0.75, 1, 1.5, 2, 2.5, 3grams) and at least (or at least about) 0.70 g (e.g. at least about 0.7,0.75, 1, 1.5, 2, 2.5, 3 grams). In some embodiments, a compositionfurther comprises a PDE inhibitor in a synergizing amount. In someembodiments, a composition further comprises a sirtuin pathway activatorin a synergizing amount. In some embodiments, resveratrol is replacedwith a PDE inhibitor or other sirtuin pathway activator in a synergizingamount. In compositions comprising a PDE inhibitor or methods comprisingadministration of a PDE inhibitor (separately from or concurrently withone or more other components), the PDE inhibitor may be provided in anamount that produces a peak plasma concentration of about, less thanabout, or more than about 0.1, 1, 5, 10, 25, 50, 100, 500, 1000, 2500,5000, 10000, or more nM.

Another embodiment provides for a composition containing synergizingamounts of HMB, leucine and resveratrol. In such compositions, the totalamount of leucine and HMB within the composition can be less than 3.0 g(or less than about 3.0 g; e.g. less than about 0.7, 0.75, 1, 1.5, 2,2.5, 3 grams) and at least 0.70 g (or at least about 0.70 g; e.g. atleast about 0.7, 0.75, 1, 1.5, 2, 2.5, 3 grams). Compositions containingboth leucine and HMB can contain amounts of leucine and HMB that totalabout, less than about, or more than about 0.70 g to 3.0 g (about 0.70 gto about 3.0 g; e.g. 0.7, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, or more grams),0.75 g to 3.0 g (about 0.75 g to about 3.0 g), or 1.0 g to 3.0 g (about1.0 g to about 3.0 g) within the composition and resveratrol insynergizing amounts (at least 35 mg of resveratrol and no more than 500(or about 500) mg resveratrol (e.g. about, less than about, or more thanabout 35, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mgresveratrol) or an amount of resveratrol between 50 and 500 mg (or about50 to about 500 mg). In some embodiments, a composition furthercomprises a PDE inhibitor in a synergizing amount. In some embodiments,a composition further comprises a sirtuin pathway activator in asynergizing amount. In some embodiments, resveratrol is replaced with aPDE inhibitor or a sirtuin pathway activator in a synergizing amount. Incompositions comprising a PDE inhibitor or methods comprisingadministration of a PDE inhibitor (separately from or concurrently withone or more other components), the PDE inhibitor may be provided in anamount that produces a peak plasma concentration of about, less thanabout, or more than about 0.1, 1, 5, 10, 25, 50, 100, 500, 1000, 2500,5000, 10000, or more nM.

Yet another embodiment provides for a composition containing synergizingamounts of HMB, leucine, KIC and resveratrol. In such compositions, thetotal amount of leucine, KIC and HMB within the composition can be lessthan 3.0 g (or less than about 3.0 g; e.g. less than about 0.7, 0.75, 1,1.5, 2, 2.5, 3 grams) and at least 0.70 g (or at least about 0.70 g;e.g. at least about 0.7, 0.75, 1, 1.5, 2, 2.5, 3 grams). Thus,compositions containing leucine, KIC and HMB can contain amounts ofleucine, KIC and HMB that total about, less than about, or more thanabout 0.70 g to 3.0 g (about 0.70 g to about 3.0 g; e.g. 0.7, 0.75, 1,1.5, 2, 2.5, 3 grams), 0.75 g to 3.0 g (about 0.75 g to about 3.0 g), or1.0 g to 3.0 g (about 1.0 g to about 3.0 g) within the composition andresveratrol in synergizing amounts (at least 35 mg of resveratrol and nomore than 500 (or about 500) mg resveratrol (e.g. about, less thanabout, or more than about 35, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, or 500 mg resveratrol) or an amount of resveratrol between 50 and500 mg (or about 50 to about 500 mg). In some embodiments, a compositionfurther comprises a PDE inhibitor in a synergizing amount. In someembodiments, a composition further comprises a sirtuin pathway activatorin a synergizing amount. In some embodiments, resveratrol is replacedwith a PDE inhibitor or a sirtuin pathway activator in a synergizingamount. In compositions comprising a PDE inhibitor or methods comprisingadministration of a PDE inhibitor (separately from or concurrently withone or more other components), the PDE inhibitor may be provided in anamount that produces a peak plasma concentration of about, less thanabout, or more than about 0.1, 1, 5, 10, 25, 50, 100, 500, 1000, 2500,5000, 10000, or more nM.

Still other embodiments provide compositions comprising: a) about, lessthan about, or more than about 50 to 100 mg resveratrol (e.g. 50, 60,70, 80, 90, 100, or more mg) and about, less than about, or more thanabout 400 mg to 500 mg HMB (e.g. 400, 425, 450, 475, 500, or more mg);b) about, less than about, or more than about 50 to 100 mg resveratrol(e.g. 50, 60, 70, 80, 90, 100, or more mg) and about, less than about,or more than about 750 mg to 1250 mg leucine (e.g. 750, 850, 950, 1050,1150, 1250 or more mg); c) about, less than about, or more than about 50to 100 mg resveratrol (e.g. 50, 60, 70, 80, 90, 100, or more mg) andabout, less than about, or more than about 750 mg to 1250 mg KIC (e.g.750, 850, 950, 1050, 1150, 1250 or more mg); or d) about, less thanabout, or more than about 50 mg to about 100 mg resveratrol (e.g. 50,60, 70, 80, 90, 100, or more mg) and: i) a combination of HMB and KIC inan amount of about 400 mg and about 1250 mg (e.g. 400, 500, 600, 700,800, 900, 1000, 1100, 1250, or more mg); ii) a combination of HMB andleucine in an amount of about 400 mg and about 1250 mg (e.g. 400, 500,600, 700, 800, 900, 1000, 1100, 1250, or more mg); iii) a combination ofKIC and leucine in an amount of about 400 mg and about 1250 mg (e.g.400, 500, 600, 700, 800, 900, 1000, 1100, 1250, or more mg); or iv) acombination of HMB, KIC and leucine in an amount of about 400 mg andabout 1250 mg (e.g. 400, 500, 600, 700, 800, 900, 1000, 1100, 1250, ormore mg). In some embodiments, a composition further comprises a PDEinhibitor in a synergizing amount. In some embodiments, a compositionfurther comprises a sirtuin pathway activator in a synergizing amount.In some embodiments, resveratrol is replaced with a PDE inhibitor or asirtuin pathway activator in a synergizing amount. In compositionscomprising a PDE inhibitor or methods comprising administration of a PDEinhibitor (separately from or concurrently with one or more othercomponents), the PDE inhibitor may be provided in an amount thatproduces a peak plasma concentration of about, less than about, or morethan about 0.1, 1, 5, 10, 25, 50, 100, 500, 1000, 2500, 5000, 10000, ormore nM.

In some embodiments a unit dosage can comprise resveratrol incombination with one or more other components. In some embodiments, aunit dosage comprises one or more of: about, less than about, or morethan about 50, 100, 200, 300, 400, 500 or more mg of HMB; about, lessthan about, or more than about 10, 20, 30, 40, 50, 75, 100, or more mgresveratrol; about, less than about, or more than about 2.5, 5, 7.5, 10,12.5, 15, 17.5, 20, or more mg of vitamin B6; about, less than about, ormore than about 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, or more μg ofvitamin D; about, less than about, or more than about 5, 10, 25, 50, 75,100, 150, 200, or more μg of vitamin K2; and about, less than about, ormore than about 400, 500, 600, 700, 800, 900, 1000, 1100, 1250, 1500, ormore mg of leucine. A unit dosage can comprise about, less than about,or more than about 500 mg beta hydroxyl, beta methyl butyrate and about,less than about, or more than about 50 mg resveratrol. A unit dosage cancomprise about, less than about, or more than about 500 mg beta hydroxy,beta methyl butyrate; and about, less than about, or more than about 50mg resveratrol; and about, less than about, or more than about 15 mgvitamin B6. A unit dosage can comprise about, less than about, or morethan about 1.125 g leucine and about, less than about, or more thanabout 50 mg resveratrol. A unit dosage can comprise about, less thanabout, or more than about 1.125 g leucine, 50 mg resveratrol and 15 mgvitamin B6. A unit dosage can comprise about, less than about, or morethan about 750 mg leucine, 35 mg resveratrol and 10 mg vitamin B6. Aunit dosage may comprise about 500 mg HMB, 51 mg resveratrol (98%), 12.5μg of vitamin D, and 50 μg of vitamin K2. In some embodiments, acomposition further comprises a PDE inhibitor in a synergizing amount.In some embodiments, a composition further comprises a sirtuin pathwayactivator in a synergizing amount. In some embodiments, resveratrol isreplaced with a PDE inhibitor or a sirtuin pathway activator in asynergizing amount. In compositions comprising a PDE inhibitor ormethods comprising administration of a PDE inhibitor (separately from orconcurrently with one or more other components), the PDE inhibitor maybe provided in an amount that produces a peak plasma concentration ofabout, less than about, or more than about 0.1, 1, 5, 10, 25, 50, 100,500, 1000, 2500, 5000, 10000, or more nM.

In some embodiments a unit dosage can comprise cholorogenic acid (e.g.about, less than about, or more than about 25, 50, 75, 100, 150, 200, ormg) in combination with one or more other components in about, less thanabout, or more than about the indicated amounts. A unit dosage cancomprise 500 mg beta hydroxy, beta methyl butyrate (e.g. 50, 100, 200,300, 400, 500 or more mg) and 100 mg chlorogenic acid. A unit dosage cancomprise 500 mg beta hydroxy, beta methyl butyrate (e.g. 50, 100, 200,300, 400, 500 or more mg); and 100 mg chlorogenic acid; and 15 mgvitamin B6. A unit dosage can comprise 1.125 g leucine (e.g. 400, 500,600, 700, 800, 900, 1000, 1100, 1250, or more mg) and 100 mg chlorogenicacid. A unit dosage can comprise 1.125 g leucine (e.g. 400, 500, 600,700, 800, 900, 1000, 1100, 1250, or more mg); 100 mg chlorogenic acid;and 15 mg vitamin B6 (e.g. 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, or moremg). A unit dosage can comprise 750 mg leucine, 75 mg chlorogenic acidand 10 mg vitamin B6.

In some embodiments a unit dosage can comprise quinic acid in about,less than about, or more than about the indicated amounts (e.g. 10, 15,20, 25, 30, 40, 50, or more mg), in combination with one or more othercomponents in about, less than about, or more than about the indicatedamounts. A unit dosage can comprise 500 mg beta hydroxy, beta methylbutyrate (e.g. 50, 100, 200, 300, 400, 500 or more mg) and 25 mg quinicacid. A unit dosage can comprise 500 mg beta hydroxy, beta methylbutyrate (e.g. 50, 100, 200, 300, 400, 500 or more mg), 25 mg quinicacid and 15 mg vitamin B6 (e.g. 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, ormore mg). A unit dosage can comprise 1.125 g leucine (e.g. 400, 500,600, 700, 800, 900, 1000, 1100, 1250, or more mg) and 25 mg quinic acid.A unit dosage can comprise 1.125 g leucine (e.g. 400, 500, 600, 700,800, 900, 1000, 1100, 1250, or more mg), 25 mg quinic acid and 15 mgvitamin B6 (e.g. 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, or more mg). Aunit dosage can comprise 750 mg leucine, 15 mg quinic acid and 10 mgvitamin B6.

In some embodiments a unit dosage can comprise fucoxanthin in about,less than about, or more than about the indicated amounts (e.g. 0.5,0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 3, 5, or more mg) in combinationwith one or more other components in about, less than about, or morethan about the indicated amounts. A unit dosage can comprise 500 mg betahydroxy, beta methyl butyrate (e.g. 50, 100, 200, 300, 400, 500 or moremg) and 2.5 mg fucoxanthin. A unit dosage can comprise 500 mg betahydroxy, beta methyl butyrate (e.g. 50, 100, 200, 300, 400, 500 or moremg), 2.5 mg fucoxanthin and 15 mg vitamin B6 (e.g. 2.5, 5, 7.5, 10,12.5, 15, 17.5, 20, or more mg). A unit dosage can comprise 1.125 gleucine (e.g. 400, 500, 600, 700, 800, 900, 1000, 1100, 1250, or moremg) and 2.5 mg fucoxanthin. A unit dosage can comprise 1.125 g leucine(e.g. 400, 500, 600, 700, 800, 900, 1000, 1100, 1250, or more mg), 2.5mg fucoxanthin and 15 mg vitamin B6 (e.g. 2.5, 5, 7.5, 10, 12.5, 15,17.5, 20, or more mg). A unit dosage can comprise 750 mg leucine, 1.5 mgfucoxanthin and 10 mg vitamin B6.

In some embodiments, a composition comprises an amount of anantidiabetic agent, such as a biguanide (e.g. metformin). The amount ofantidiabetic agent may be a subtheratpeutic amount, and/or an amountthat is synergistic with one or more other compounds in the compositionor one or more othe comounds administered simultaneously or in closetemporal proximity with the composition. In some embodiments, theantidiabetic agent is administered in a very low dose, a low dose, amedium dose, or a high dose, which describes the relationship betweentwo doses, and generally do not define any particular dose range. Forexample, a daily very low dose of metformin may comprise about, lessthan about, or more than about 5 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg, 75mg/kg, 100 mg/kg, or more; a daily low dose of metformin may compriseabout, less than about, or more than about 75 mg/kg, 100 mg/kg, 150mg/kg, 175 mg/kg, 200 mg/kg, or more; a daily medium dose of metforminmay comprise about, less than about, or more than about 150 mg/kg, 175mg/kg, 200 mg/kg, 250 mg/kg, 300; and a daily high dose of metformin maycomprise about, less than about, or more than about 200 mg/kg, 250mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 500 mg/kg, 700 mg/kg, or more.

In some embodiments a unit dosage can comprise metformin in about, lessthan about, or more than about the indicated amounts (e.g. 25, 50, 100,150, 200, 250, 300, 400, 500, or more mg) in combination with one ormore other components in about, less than about, or more than about theindicated amounts (such as 10, 20, 30, 40, 50, 75, 100, or more mg ofresveratrol; 50, 100, 200, 300, 400, 500 or more mg HMB; and/or 400,500, 600, 700, 800, 900, 1000, 1100, 1250, or more mg of leucine). Aunit dosage can comprise about, less than about or more than about 50 mgmetformin, 500 mg beta hydroxy, beta methyl butyrate and 50 mgresveratrol. A unit dosage can comprise about, less than about or morethan about 50 mg metformin, 1.125 g leucine and 50 mg resveratrol. Aunit dosage can comprise about, less than about or more than about 100mg metformin, 500 mg beta hydroxy, beta methyl butyrate and 50 mgresveratrol. A unit dosage can comprise about, less than about or morethan about 100 mg metformin, 1.125 g leucine and 50 mg resveratrol. Aunit dosage can comprise about, less than about or more than about 250mg metformin, 500 mg beta hydroxy, beta methyl butyrate and 50 mgresveratrol. A unit dosage can comprise about, less than about or morethan about 250 mg metformin, 1.125 g leucine and 50 mg resveratrol. Insome embodiments, a composition further comprises a PDE inhibitor in asynergizing amount. In some embodiments, a metformin composition furthercomprises a sirtuin pathway activator in a synergizing amount. In someembodiments, resveratrol in an example composition is replaced with aPDE inhibitor or a sirtuin pathway activator in a synergizing amount. Incompositions comprising a PDE inhibitor or methods comprisingadministration of a PDE inhibitor (separately from or concurrently withone or more other components), the PDE inhibitor may be provided in anamount that produces a peak plasma concentration of about, less thanabout, or more than about 0.1, 1, 5, 10, 25, 50, 100, 500, 1000, 2500,5000, 10000, or more nM.

In some embodiments of the invention, the combination compositions canhave a specified ratio of branched chain amino acids and/or metabolitesthereof to a sirtuin pathway activator. The specified ratio can providefor effective and/or synergistic regulation of energy metabolism. Forexample, the specified ratio can cause a decrease in weight gain of asubject, a decrease in visceral adipose volume of a subject, an increasein fat oxidation of a subject, an increase in insulin sensitivity of asubject, an increase of glucose uptake in muscle of a subject, adecrease in inflammation markers, an increase in vasodilatation, and/oran increase in body temperature. Such beneficial effects can resultfrom, in part, an increase in mitochondrial biogenesis, or a variety ofother changes in the energy metabolism pathway. The ratio of branchedchain amino acids and/or metabolites thereof to a sirtuin pathwayactivator can be a mass ratio, a molar ratio, or a volume ratio.

In some embodiments, the molar ratio of (a) branched chain amino acidsand/or metabolites thereof to (b) a sirtuin pathway activator is aboutor greater than about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 100, 120, or 150. In other embodiments, the molar ratio ofone or more branched chain amino acids and/or metabolites thereof tosirtuin pathway activator contained in the subject compositions is aboutor greater than about 20, 30, 40, 50, 60, 70, 80, 90, 95, 90, 95, 100,105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, 300,350, 400, or 500. In some embodiments, the molar ratio of component (a)to (b) in said composition is greater than about 20, 40, 60, 80, 100,120, or 150. In some embodiments, the molar ratio of component (a) to(b) in said composition is greater than about 80, 100, 120, or 150. Insome embodiments, the molar ratio of component (a) to (b) in saidcomposition is greater than about 80, 100, 120, or 150. In someembodiments, the molar ratio of component (a) to (b) in said compositionis greater than about 200, 250, or 300. In some embodiments, the molarratio of component (a) to (b) in said composition is greater than about40, 150, 250, or 500.

In some embodiments, the molar or mass ratios are circulating molar ormass ratios achieved after administration one or more compositions to asubject. The compositions can be a combination composition describedherein. The molar ratio of a combination composition in a dosing formcan be adjusted to achieve a desired circulating molar ratio. The molarratio can be adjusted to account for the bioavailiability, the uptake,and the metabolic processing of the one or more components of acombination composition. For example, if the bioavailiability of acomponent is low, then the molar amount of a that component can beincreased relative to other components in the combination composition.In some embodiments, the circulating molar or mass ratio is achievedwithin about 0.1, 0.5, 0.75, 1, 3, 5, or 10, 12, 24, or 48 hours afteradministration. The circulating molar or mass ratio can be maintainedfor a time period of about or greater than about 0.1, 1, 2, 5, 10, 12,18, 24, 36, 48, 72, or 96 hours.

In some embodiments, the circulating molar ratio of leucine toresveratrol (or sirtuin pathway activator) is about or greater thanabout 1000, 1500, 2000, 2500, 3000, 3500, 4000, 10000, 50000, or more.In some embodiments, the mass ratio of leucine to resveratrol is aboutor greater than about 750, 1000, 1200, 1500, 1700, 2000, or 2500.

The circulating molar ratio of HMB to resveratrol (or sirtuin pathwayactivator) can be about or greater than about 3, 5, 10, 15, 20, 25, 30,35, 40, 50, 60, 75, 100, 250, 500, or more. In some embodiments, themass ratio of HMB to resveratrol is about or greater than about 1, 3, 6,9, 12, 15, 20, or 25.

In some embodiments, the circulating mass ratio of HMB to resveratrol(or sirtuin pathway activator) is about or greater than about 100, 120,140, 160, 180, 200, 220, or 250. In some embodiments, the mass ratio ofHMB to resveratrol is about or greater than about 400, 600, 800, 1000,1200, or 1400.

In some embodiments, the circulating molar ratio of HMB to chlorogenicacid is about or greater than about 5, 10, 20, or 40. In someembodiments, the molar ratio of leucine to chlorogenic acid is about orgreater than about 500, 1000, 2000, or 4000.

In some embodiments, the circulating molar ratio of HMB to caffeic acidis about or greater than about 2, 5, 10, or 20. In some embodiments, themolar ratio of leucine to caffeic acid is about or greater than about200, 500, 1000, or 2000.

In some embodiments, the circulating molar ratio of HMB to quinic acidis about or greater than about 5, 10, 20, or 40. In some embodiments,the molar ratio of leucine to quinic acid is about or greater than about500, 1000, 2000, or 4000.

In some embodiments, the circulating molar ratio of HMB to cinnamic acidis about or greater than about 5, 10, 20, or 40. In some embodiments,the molar ratio of leucine to cinnamic acid is about or greater thanabout 500, 1000, 2000, or 4000.

In some embodiments, the circulating molar ratio of HMB to ferulic acidis about or greater than about 5, 10, 20, or 40. In some embodiments,the molar ratio of leucine to ferulic acid is about or greater thanabout 500, 1000, 2000, or 4000.

In some embodiments, the circulating molar ratio of HMB to piceatannolis about or greater than about 2000, 5000, 10000, or 20000. In someembodiments, the molar ratio of leucine to piceatannol is about orgreater than about 200000, 500000, 1000000, or 2000000.

In some embodiments, the circulating molar ratio of HMB to ellagic acidis about or greater than about 0.05, 0.1, 0.2, or 0.4. In someembodiments, the molar ratio of leucine to ellagic acid is about orgreater than about 5, 10, 20, or 40.

In some embodiments, the circulating molar ratio of HMB toepigallocatechin gallate is about or greater than about 2, 5, 10, or 20.In some embodiments, the molar ratio of leucine to epigallocatechingallate is about or greater than about 200, 500, 1000, or 2000.

In some embodiments, the circulating molar ratio of HMB to fucoxanthinis about or greater than about 20, 50, 100, or 200. In some embodiments,the molar ratio of leucine to fucoxanthin is about or greater than about2000, 5000, 10000, or 20000.

In some embodiments, the circulating mass ratio of HMB to grape seedextract is about or greater than about 0.3, 0.6, 1.2, or 2.4. In someembodiments, the mass ratio of leucine to grape seed extract is about orgreater than about 30, 65, 130, or 260.

In some embodiments, the circulating molar ratio of HMB to metformin isabout or greater than about 0.02, 0.05, 0.1, or 0.2. In someembodiments, the molar ratio of leucine to metformin is about or greaterthan about 2, 5, 10, or 20

In some embodiments, the circulating molar ratio of HMB to rosiglitazoneis about or greater than about 10, 25, 50, or 100. In some embodiments,the molar ratio of leucine to rosiglitazone is about or greater thanabout 1000, 2500, 5000, or 10000.

Dosing Forms

The compositions described herein can be compounded into a variety ofdifferent dosage forms. It can be used orally as a tablet, chewabletablet, caplets, capsule, soft gelatin capsules, lozenges or solution.It can also be used as a nasal spray or for injection when in itssolution form. In some embodiments, the composition may be a liquidcomposition suitable for oral consumption. Compositions of the inventionsuitable for oral administration can be presented as discrete dosageforms, such as capsules, cachets, or tablets, or liquids or aerosolsprays each containing a predetermined amount of an active ingredient asa powder or in granules, a solution, or a suspension in an aqueous ornon-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquidemulsion, including liquid dosage forms (e.g., a suspension or slurry),and oral solid dosage forms (e.g., a tablet or bulk powder). Oral dosageforms may be formulated as tablets, pills, dragees, capsules, emulsions,lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by an individual or apatient to be treated. Such dosage forms can be prepared by any of themethods of formulation. For example, the active ingredients can bebrought into association with a carrier, which constitutes one or morenecessary ingredients. Capsules suitable for oral administration includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. Optionally, theinventive composition for oral use can be obtained by mixing acomposition a solid excipient, optionally grinding a resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). In general, the compositions are prepared byuniformly and intimately admixing the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired presentation. Forexample, a tablet can be prepared by compression or molding, optionallywith one or more accessory ingredients. Compressed tablets can beprepared by compressing in a suitable machine the active ingredient in afree-flowing form such as powder or granules, optionally mixed with anexcipient such as, but not limited to, a binder, a lubricant, an inertdiluent, and/or a surface active or dispersing agent. Molded tablets canbe made by molding in a suitable machine a mixture of the powderedcompound moistened with an inert liquid diluent.

The liquid forms, in which the formulations disclosed herein may beincorporated for administration orally or by injection, include aqueoussolution, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil, or peanut oil as well as elixirs and similar pharmaceuticalvehicles. Suitable dispersing or suspending agents for aqueoussuspensions include synthetic natural gums, such as tragacanth, acacia,alginate, dextran, sodium carboxymethyl cellulose, methylcellulose,polyvinylpyrrolidone or gelatin.

A subject can be treated by combination of an injectable composition andan orally ingested composition.

Liquid preparations for oral administration may take the form of, forexample, solutions, syrups or suspensions, or they may be presented as adry product for reconstitution with water or other suitable vehiclesbefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, methyl cellulose or hydrogenated ediblefats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters or ethyl alcohol); preservatives(e.g., methyl or propyl p-hydroxybenzoates or sorbic acid); andartificial or natural colors and/or sweeteners.

The preparation of pharmaceutical compositions of this invention isconducted in accordance with generally accepted procedures for thepreparation of pharmaceutical preparations. See, for example,Remington's Pharmaceutical Sciences 18th Edition (1990), E. W. Martined., Mack Publishing Co., PA. Depending on the intended use and mode ofadministration, it may be desirable to process the magnesium-counter ioncompound further in the preparation of pharmaceutical compositions.Appropriate processing may include mixing with appropriate non-toxic andnon-interfering components, sterilizing, dividing into dose units, andenclosing in a delivery device.

This invention further encompasses anhydrous compositions and dosageforms comprising an active ingredient, since water can facilitate thedegradation of some compounds. For example, water may be added (e.g.,5%) in the arts as a means of simulating long-term storage in order todetermine characteristics such as shelf-life or the stability offormulations over time Anhydrous compositions and dosage forms of theinvention can be prepared using anhydrous or low moisture containingingredients and low moisture or low humidity conditions. Compositionsand dosage forms of the invention which contain lactose can be madeanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected. An anhydrouscomposition may be prepared and stored such that its anhydrous nature ismaintained. Accordingly, anhydrous compositions may be packaged usingmaterials known to prevent exposure to water such that they can beincluded in suitable formulary kits. Examples of suitable packaginginclude, but are not limited to, hermetically sealed foils, plastic orthe like, unit dose containers, blister packs, and strip packs.

An ingredient described herein can be combined in an intimate admixturewith a pharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier can take a wide variety of formsdepending on the form of preparation desired for administration. Inpreparing the compositions for an oral dosage form, any of the usualpharmaceutical media can be employed as carriers, such as, for example,water, glycols, oils, alcohols, flavoring agents, preservatives,coloring agents, and the like in the case of oral liquid preparations(such as suspensions, solutions, and elixirs) or aerosols; or carrierssuch as starches, sugars, micro-crystalline cellulose, diluents,granulating agents, lubricants, binders, and disintegrating agents canbe used in the case of oral solid preparations, in some embodimentswithout employing the use of lactose. For example, suitable carriersinclude powders, capsules, and tablets, with the solid oralpreparations. If desired, tablets can be coated by standard aqueous ornonaqueous techniques.

Some examples of materials which may serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

Binders suitable for use in dosage forms include, but are not limitedto, corn starch, potato starch, or other starches, gelatin, natural andsynthetic gums such as acacia, sodium alginate, alginic acid, otheralginates, powdered tragacanth, guar gum, cellulose and its derivatives(e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulosecalcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methylcellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose,microcrystalline cellulose, and mixtures thereof.

Lubricants which can be used to form compositions and dosage forms ofthe invention include, but are not limited to, calcium stearate,magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol,mannitol, polyethylene glycol, other glycols, stearic acid, sodiumlauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil,cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethylaureate, agar, ormixtures thereof. Additional lubricants include, for example, a syloidsilica gel, a coagulated aerosol of synthetic silica, or mixturesthereof. A lubricant can optionally be added, in an amount of less thanabout 1 weight percent of the composition.

Lubricants can be also be used in conjunction with tissue barriers whichinclude, but are not limited to, polysaccharides, polyglycans,seprafilm, interceed and hyaluronic acid.

Disintegrants may be used in the compositions of the invention toprovide tablets that disintegrate when exposed to an aqueousenvironment. Too much of a disintegrant may produce tablets which maydisintegrate in the bottle. Too little may be insufficient fordisintegration to occur and may thus alter the rate and extent ofrelease of the active ingredient(s) from the dosage form. Thus, asufficient amount of disintegrant that is neither too little nor toomuch to detrimentally alter the release of the active ingredient(s) maybe used to form the dosage forms of the compounds disclosed herein. Theamount of disintegrant used may vary based upon the type of formulationand mode of administration, and may be readily discernible to those ofordinary skill in the art. About 0.5 to about 15 weight percent ofdisintegrant, or about 1 to about 5 weight percent of disintegrant, maybe used in the pharmaceutical composition. Disintegrants that can beused to form compositions and dosage forms of the invention include, butare not limited to, agar-agar, alginic acid, calcium carbonate,microcrystalline cellulose, croscarmellose sodium, crospovidone,polacrilin potassium, sodium starch glycolate, potato or tapioca starch,other starches, pre-gelatinized starch, other starches, clays, otheralgins, other celluloses, gums or mixtures thereof.

Examples of suitable fillers for use in the compositions and dosageforms disclosed herein include, but are not limited to, talc, calciumcarbonate (e.g., granules or powder), microcrystalline cellulose,powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol,starch, pre-gelatinized starch, and mixtures thereof.

When aqueous suspensions and/or elixirs are desired for oraladministration, the active ingredient therein may be combined withvarious sweetening or flavoring agents, coloring matter or dyes and, ifso desired, emulsifying and/or suspending agents, together with suchdiluents as water, ethanol, propylene glycol, glycerin and variouscombinations thereof.

The tablets can be uncoated or coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate canbe employed. Formulations for oral use can also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin, or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, for example, peanut oil, liquidparaffin or olive oil.

In one embodiment, the composition may include a solubilizer to ensuregood solubilization and/or dissolution of the compound of the presentinvention and to minimize precipitation of the compound of the presentinvention. This can be especially important for compositions fornon-oral use, e.g., compositions for injection. A solubilizer may alsobe added to increase the solubility of the hydrophilic drug and/or othercomponents, such as surfactants, or to maintain the composition as astable or homogeneous solution or dispersion.

The composition can further include one or more pharmaceuticallyacceptable additives and excipients. Such additives and excipientsinclude, without limitation, detackifiers, anti-foaming agents,buffering agents, polymers, antioxidants, preservatives, chelatingagents, viscomodulators, tonicifiers, flavorants, colorants, odorants,opacifiers, suspending agents, binders, fillers, plasticizers,lubricants, and mixtures thereof. A non-exhaustive list of examples ofexcipients includes monoglycerides, magnesium stearate, modified foodstarch, gelatin, microcrystalline cellulose, glycerin, stearic acid,silica, yellow beeswax, lecithin, hydroxypropylcellulose, croscarmellosesodium, and crospovidone.

The compositions described herein can also be formulated asextended-release, sustained-release or time-release such that one ormore components are released over time. Delayed release can be achievedby formulating the one or more components in a matrix of a variety ofmaterials or by microencapsulation. The compositions can be formulatedto release one or more components over a time period of 4, 6, 8, 12, 16,20, or 24 hours. The release of the one or more components can be at aconstant or changing rate.

Using the controlled release dosage forms provided herein, the one ormore cofactors can be released in its dosage form at a slower rate thanobserved for an immediate release formulation of the same quantity ofcomponents. In some embodiments, the rate of change in the biologicalsample measured as the change in concentration over a defined timeperiod from administration to maximum concentration for an controlledrelease formulation is less than about 80%, 70%, 60%, 50%, 40%, 30%,20%, or 10% of the rate of the immediate release formulation.Furthermore, in some embodiments, the rate of change in concentrationover time is less than about 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%of the rate for the immediate release formulation.

In some embodiments, the rate of change of concentration over time isreduced by increasing the time to maximum concentration in a relativelyproportional manner. For example, a two-fold increase in the time tomaximum concentration may reduce the rate of change in concentration byapproximately a factor of 2. As a result, the one or more cofactors maybe provided so that it reaches its maximum concentration at a rate thatis significantly reduced over an immediate release dosage form. Thecompositions of the present invention may be formulated to provide ashift in maximum concentration by 24 hours, 16 hours, 8 hours, 4 hours,2 hours, or at least 1 hour. The associated reduction in rate of changein concentration may be by a factor of about 0.05, 0.10, 0.25, 0.5 or atleast 0.8. In certain embodiments, this is accomplished by releasingless than about 30%, 50%, 75%, 90%, or 95% of the one or more cofactorsinto the circulation within one hour of such administration.

Optionally, the controlled release formulations exhibit plasmaconcentration curves having initial (e.g., from 2 hours afteradministration to 4 hours after administration) slopes less than 75%,50%, 40%, 30%, 20% or 10% of those for an immediate release formulationof the same dosage of the same cofactor.

In some embodiments, the rate of release of the cofactor as measured indissolution studies is less than about 80%, 70%, 60% 50%, 40%, 30%, 20%,or 10% of the rate for an immediate release formulation of the samecofactor over the first 1, 2, 4, 6, 8, 10, or 12 hours.

The controlled release formulations provided herein can adopt a varietyof formats. In some embodiments, the formulation is in an oral dosageform, including liquid dosage forms (e.g., a suspension or slurry), andoral solid dosage forms (e.g., a tablet or bulk powder), such as, butnot limited to those, those described herein.

The controlled release tablet of a formulation disclosed herein can beof a matrix, reservoir or osmotic system. Although any of the threesystems is suitable, the latter two systems can have more optimalcapacity for encapsulating a relatively large mass, such as for theinclusion of a large amount of a single cofactor, or for inclusion of aplurality of cofactors, depending on the genetic makeup of theindividual. In some embodiments, the slow-release tablet is based on areservoir system, wherein the core containing the one or more cofactorsis encapsulated by a porous membrane coating which, upon hydration,permits the one or more cofactors to diffuse through. Because thecombined mass of the effective ingredients is generally in gramquantity, an efficient delivery system can provide optimal results.

Thus, tablets or pills can also be coated or otherwise compounded toprovide a dosage form affording the advantage of prolonged action. Forexample, the tablet or pill can comprise an inner dosage an outer dosagecomponent, the latter being in the form of an envelope over the former.The two components can be separated by an enteric layer which serves toresist disintegration in the stomach and permits the inner component topass intact into the duodenum or to be delayed in release. A variety ofmaterials can be used for such enteric layers or coatings such materialsincluding a number of polymeric acids and mixtures of polymeric acidswith such materials as shellac, cetyl alcohol and cellulose acetate. Insome embodiments, a formulation comprising a plurality of cofactors mayhave different cofactors released at different rates or at differenttimes. For example, there can be additional layers of cofactorsinterspersed with eneteric layers.

Methods of making sustained release tablets are known in the art, e.g.,see U.S. Patent Publications 2006/051416 and 2007/0065512, or otherreferences disclosed herein. Methods such as described in U.S. Pat. Nos.4,606,909, 4,769,027, 4,897,268, and 5,395,626 can be used to preparesustained release formulations of the one or more cofactors determinedby the genetic makeup of an individual. In some embodiments, theformulation is prepared using OROS® technology, such as described inU.S. Pat. Nos. 6,919,373, 6,923,800, 6,929,803, and 6,939,556. Othermethods, such as described in U.S. Pat. Nos. 6,797,283, 6,764,697, and6,635,268, can also be used to prepare the formulations disclosedherein.

In some embodiments, the compositions can be formulated in a foodcomposition. For example, the compositions can be a beverage or otherliquids, solid food, semi-solid food, with or without a food carrier.For example, the compositions can include a black tea supplemented withany of the compositions described herein. The composition can be a dairyproduct supplemented any of the compositions described herein. In someembodiments, the compositions can be formulated in a food composition.For example, the compositions can comprise a beverage, solid food,semi-solid food, or a food carrier.

In some embodiments, liquid food carriers, such as in the form ofbeverages, such as supplemented juices, coffees, teas, sodas, flavoredwaters, and the like can be used. For example, the beverage can comprisethe formulation as well as a liquid component, such as various deodorantor natural carbohydrates present in conventional beverages. Examples ofnatural carbohydrates include, but are not limited to, monosaccharidessuch as, glucose and fructose; disaccharides such as maltose andsucrose; conventional sugars, such as dextrin and cyclodextrin; andsugar alcohols, such as xylitol and erythritol. Natural deodorant suchas taumatin, stevia extract, levaudioside A, glycyrrhizin, and syntheticdeodorant such as saccharin and aspartame may also be used. Agents suchas flavoring agents, coloring agents, and others can also be used. Forexample, pectic acid and the salt thereof, alginic acid and the saltthereof, organic acid, protective colloidal adhesive, pH controllingagent, stabilizer, a preservative, glycerin, alcohol, or carbonizingagents can also be used. Fruit and vegetables can also be used inpreparing foods or beverages comprising the formulations discussedherein.

Alternatively, the compositions can be a snack bar supplemented with anyof the compositions described herein. For example, the snack bar can bea chocolate bar, a granola bar, or a trail mix bar. In yet anotherembodiment, the present dietary supplement or food compositions areformulated to have suitable and desirable taste, texture, and viscosityfor consumption. Any suitable food carrier can be used in the presentfood compositions. Food carriers of the present invention includepractically any food product. Examples of such food carriers include,but are not limited to food bars (granola bars, protein bars, candybars, etc.), cereal products (oatmeal, breakfast cereals, granola,etc.), bakery products (bread, donuts, crackers, bagels, pastries,cakes, etc.), beverages (milk-based beverage, sports drinks, fruitjuices, alcoholic beverages, bottled waters), pastas, grains (rice,corn, oats, rye, wheat, flour, etc.), egg products, snacks (candy,chips, gum, chocolate, etc.), meats, fruits, and vegetables. In anembodiment, food carriers employed herein can mask the undesirable taste(e.g., bitterness). Where desired, the food composition presented hereinexhibit more desirable textures and aromas than that of any of thecomponents described herein. For example, liquid food carriers may beused according to the invention to obtain the present food compositionsin the form of beverages, such as supplemented juices, coffees, teas,and the like. In other embodiments, solid food carriers may be usedaccording to the invention to obtain the present food compositions inthe form of meal replacements, such as supplemented snack bars, pasta,breads, and the like. In yet other embodiments, semi-solid food carriersmay be used according to the invention to obtain the present foodcompositions in the form of gums, chewy candies or snacks, and the like.

The dosing of the combination compositions can be administered about,less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore times a daily. A subject can receive dosing for a period of about,less than about, or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 or more days, weeks or months. A unit dose can be afraction of the daily dose, such as the daily dose divided by the numberof unit doses to be administered per day. A unit dose can be a fractionof the daily dose that is the daily dose divided by the number of unitdoses to be administered per day and further divided by the number ofunit doses (e.g. tablets) per administration. The number of unit dosesper administration may be about, less than about, or more than about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The number of doses per day may beabout, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more. The number of unit doses per day may be determined bydividing the daily dose by the unit dose, and may be about, less thanabout, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 6, 17, 18, 19, 20, or more unit doses per day. For example, a unitdose can be about ½, ⅓, ¼, ⅕, ⅙, 1/7, ⅛, 1/9, 1/10. A unit dose can beabout one-third of the daily amount and administered to the subjectthree times daily. A unit dose can be about one-half of the daily amountand administered to the subject twice daily. A unit dose can be aboutone-fourth of the daily amount with two unit doses administered to thesubject twice daily. In some embodiments, a unit dose comprises about,less than about, or more than about 50 mg resveratrol. In someembodiments, a unit dose comprises about, less than about, or more thanabout 550 mg leucine. In some embodiments, a unit dose comprises about,less than about, or more than about 200 mg of one or more leucinemetabolites. In some embodiments, a unit dose (e.g. a unit dosecomprising leucine) is administered as two unit doses two times per day.In some embodiments, a unit dose (e.g. a unit dose comprising one ormore leucine metabolites, such as HMB) is administered as one unit dosetwo timer per day.

Compositions disclosed herein can further comprise a flavorant and canbe a solid, liquid, gel or emulsion.

Methods

The subject application provides methods of increasing sirtuin pathwayoutput (including AMPK, a signaling molecule in the sirtuin pathway) ina subject. As described herein, the output of the sirtuin pathway can becharacterized at the molecular level or by a resulting physiologicaleffect. In some embodiments, the invention provides for methods ofincreasing fatty acid oxidation in a subject comprising theadministration of a composition as disclosed herein to the subject. Invarious embodiments of the invention, a composition is administered tothe subject in an amount that delivers synergizing amounts of one ormore branched amino acids and a polyphenol sufficient to increase fattyacid oxidation within the cells of the subject.

The methods described herein can be useful for a variety ofapplications. These applications include (a) an increase insirtuin-pathway output, (b) an increase in mitochondrial biogenesis, (c)an increase in the formation of new mitochondria, (d) an increase inmitochondrial functions, (e) an increase in fatty acid oxidation, (f) anincrease in heat generation, (g) an increase in insulin sensitivity, (h)an increase in glucose uptake, (i) an increase in vasodilation, (j) adecrease in weight, (k) a decrease in adipose volume, (l) a decrease ininflammatory response or markers in a subject, and (m) an increase inirisin production. Any of these applications can be achieved byadministering one or more compositions described herein.

Accordingly, the invention provides a method for administering acomposition comprising (a) one or more types of branched amino acidsand/or metabolites thereof and (b) a sirtuin-pathway activator presentin a sub-therapeutic amount, wherein the composition is synergisticallyeffective in increasing the sirtuin-pathway output by at least about 5fold as compared to that of component (b) when it is being used alone.

The output of the pathways can be measured using one or more methods,disclosed herein and/or known in the art. For example, fatty acidoxidation can be determined by measuring oxygen consumption, or³H-labeled palmitate oxidation. Mitochondrial biogenesis can be measuredusing a mitochondrial probe by using fluorescence. AMPK activity can bedetermined by measuring AMPK phosphorylation via an ELISA assay or byWestern blot. Sirt1 activity can be determined by measuringdeacetylation of a substrate, which can be detected using a fluorophore.

An increase in sirt1, sirt2, or sirt3 is observed by applying acorresponding substrate in a deacylation assay conducted in vitro. Thesubstrate for measuring SIRT1 activity can be any substrate known in theart (for example a peptide containing amino acids 379-382 of human p53(Arg-His-Lys-Lys[Ac]). The substrate for measuring SIRT3 activity can beany substrate known in the art (for example a peptide containing aminoacids 317-320 of human p53 (Gln-Pro-Lys-Lys[Ac])). In some instances,the increase in sirt activity in one or more assays conducted in thepresence of one or more combination compositions described hereinresults in an activity increase of at least about 1, 2, 3, 5, or 10fold, as compared to the activity measured in the presence of only onecomponent of the combination compositions. For example, the use of acombination composition comprising (a) a sirtuin pathway activator (suchas resveratrol) and (b) a branched chain amino acid or metabolitethereof (such as HMB) results in an increase in sirt3 activity by atleast about 5 fold as compared to the activity measured in the presenceof (a) or (b) alone. Also, the use of a combination compositioncomprising resveratrol and leucine results in an increase in sirt1activity that is 1.5, 2, 5 or 10 fold greater than the activity measuredin the presence of only resveratrol or leucine.

The invention provides a method for administering a compositioncomprising: (a) one or more types of branched amino acids and/ormetabolites thereof, and (b) a sirtuin-pathway activator, wherein molarratio of component (a) to (b) in said composition is greater than about20, and wherein the composition when administered to a subject in needthereof synergistically enhances mitochondrial biogenesis as measured bya decrease in weight gain of a subject, a decrease in visceral adiposevolume of a subject, an increase in fat oxidation of a subject, anincrease in insulin sensitivity of a subject, an increase of glucoseuptake in muscle of a subject, a decrease in inflammation markers, anincrease in vasodilatation, and/or an increase in body temperature.

The invention provides a method for administering a compositioncomprising: a unit dosage suitable for oral ingestion, said unit dosagecomprising: (a) one or more types of branched amino acids and/ormetabolites thereof, and (b) a substantially homogeneous population ofpolyphenol molecules, and wherein the unit dosage is effective ininducing a decrease in weight gain of a subject, a decrease in visceraladipose volume of a subject, an increase in fat oxidation of a subject,an increase in insulin sensitivity of a subject, an increase of glucoseuptake in muscle of a subject, an increase in vasodilatation, and/or anincrease in body temperature.

The invention provides a method for administering a food compositioncomprising: (a) one or more types of branched amino acids and/ormetabolites thereof; (b) a sirtuin pathway activator, wherein (a) and(b) are present in an amount that synergistically effect a decrease inweight gain of a subject, a decrease in visceral adipose volume of asubject, an increase in fat oxidation of a subject, an increase ininsulin sensitivity of a subject, an increase of glucose uptake inmuscle of a subject, an increase in vasodilatation, a decrease inoxidative stress, a decrease in inflammatory stress, and/or an increasein body temperature; and (c) a food carrier.

The invention provides a method for administering a compositioncomprising: a synergistically effective amount of (a) one or more typesof branched amino acids and/or metabolites thereof; and (b) asirtuin-pathway activator, wherein the composition is substantially freeof non-branched amino acids, wherein the combination when administeredto a subject in need thereof enhances mitochondrial biogenesis to agreater degree as compared to administering to a subject component (a)or component (b) alone, and wherein the enhanced mitochondrialbiogenesis is measured by a decrease in weight of a subject, a decreasein visceral adipose volume of a subject, an increase in fat oxidation ofa subject, an increase in insulin sensitivity of a subject, an increaseof glucose uptake in muscle of a subject, an increase in vasodilatation,a decrease in oxidative stress, a decrease in inflammatory stress,and/or an increase in body temperature.

The invention provides a method for administering a compositioncomprising: a synergistically effective amount of (a) one or more typesof branched amino acids and/or metabolites thereof; and (b) asirtuin-pathway activator, wherein the composition is substantially freeof non-branched amino acids, wherein the combination when administeredto a subject in need thereof enhances mitochondrial biogenesis to agreater degree as compared to administering to a subject component (a)or component (b) alone, and wherein the enhanced mitochondrialbiogenesis is measured by a decrease in weight of a subject, a decreasein visceral adipose volume of a subject, an increase in fat oxidation ofa subject, an increase in insulin sensitivity of a subject, an increaseof glucose uptake in muscle of a subject, an increase in vasodilatation,a decrease in oxidative stress, a decrease in inflammatory stress,and/or an increase in body temperature.

The invention provides a method for administering a compositioncomprising: (a) one or more types of branched amino acids and/ormetabolites thereof, and (b) a signaling molecule downstream of PGC1α ina sirtuin-signaling pathway.

The invention provides for a method of enhancing fat oxidation in asubject in need thereof comprising administering to the subject any ofthe compositions described herein over a time period, wherein the fatoxidation in the subject is increased over the time period. The fatoxidation can be increased by about or greater than about 5, 10, 15, 20,50, 100, 200, or 500%.

The invention provides for a method of reducing an inflammatory responsein a subject in need thereof comprising administering to the subject acomposition any of the compositions described herein over a time period,wherein the inflammatory response in the subject is reduced over thetime period. The inflammatory response can be decreased by about orgreater than about 5, 10, 15, 20, 50, or 100%.

Inflammatory marker and cytokine levels, including but not limited toIL-6, adiponectin, TNF-α and CRP levels in plasma can determined byimmune assays, such as ELISA (Assay Designs, Ann Arbor, Mich.; LincoResearch, St. Charles, Mo.; and Bioscience, San Diego, Calif.).

The invention provides for a method of increasing or maintaining bodytemperature in a subject comprising administering to the subject acomposition any of the compositions described herein over a time period,wherein the body temperature in the subject is increased over the timeperiod. The body temperature can be increased by about or greater thanabout 1, 2, 3, 4, 5, 10, 15, or 20%.

The invention provides for a method of inducing vasodilatationcomprising administering to the subject a composition of any of thecompositions described herein over a time period, wherein thevasodilation in the subject is induced over the time period. Thevasodilation of blood vessels can be increased by about or greater thanabout 1, 2, 3, 5, 10, 20, 50, or 100%. The vasodilation can be measuredby optically, by measuring vasorestriction, or by a variety of othertechniques. These techniques include the invasive forearm technique, thebrachial artery ultrasound technique, and pulse wave analysis. Methodsfor measuring vasodilation are described in Lind et al., “Evaluation offour different methods to measure endothelium-dependent vasodilation inthe human peripheral circulation,” Clinical Science 2002, 102, 561-567.

The invention provides for a method of increasing irisin production,comprising administering to the subject any of the compositionsdescribed herein, wherein irisin production in the subject increasesover a time period. In some embodiments, the increase in irisinproduction (or in an indicator providing evidence thereof) is anincrease of about, or more than about 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, or more. In someembodiments, the increase in irisin production (or in an indicatorproviding evidence thereof) is an increase of about, or more than about1-fold, 3-fold, 5-fold, 6-fold, 8-fold, 10-fold, 15-fold, 20-fold,50-fold, or more. In some embodiments, the increase in irisin productionis evidenced by an increase in FNDC5 expression (e.g. as measured frommRNA and/or protein level). In some embodiments, the increase in irisinproduction is evidenced by an increase in one or more indicia of fatcell browning (e.g. fatty acid oxidation, and/or an increase inexpression of one or more brown fat selective genes in adipose tissue).Non-limiting examples of brown fat selective genes include Ucp1, Cidea,Prdm16, and Ndufs. In some embodiments, the increase in irisinproduction is evidenced by increased secretion of irisin from the cell(e.g. as measured from media in which the cell is cultured, or fromcirculating plasma in a subject). Increases in gene levels can bemeasured directly (e.g. changes in mRNA or protein levels) or indirectly(changes in effects associated with expression increase, such as anincreased expression of a downstream gene). Methods for detectingchanges in gene expression level are known in the art, and include,without limitation, methods for the detection of mRNA (e.g. RT-PCR,Northern blot, and microarray hybridization), detection of proteinproducts (e.g. Western blot, and ELISA), a detection of one or moreactivities of the translated protein (e.g. enzyme activity assays).

The invention provides for a method of treating diabetes, comprisingadministering to the subject any of the compositions described hereinover a time period, wherein the insulin sensitivity in the subject isincreased over the time period. Insulin sensitivity can be increased byabout or greater than about 1, 2, 3, 5, 10, 20, 50, 100, or 200%. Insome embodiments, a branched chain amino acid (or a metabolite thereof)and/or a sirtuin pathway activator are administered in an amount thatreduces the therapeutically effective dose of metformin for a subject.In some embodiments, the therapeutically effective dose of metformin isreduced by about or more than about 50%, 60%, 70%, 80%, 90%, 95%, 97.5%,99.9%, 99.99%, or more. In some embodiments, administration ofcompositions of the invention reduces body fat (e.g. visceral fat) byabout or more than about 5%, 10%, 15%, 20%, 25%, 50%, or more.

Insulin sensitivity can be measured using a variety of techniques,including HOMA_(IR). HOMA_(IR), which is the homeostasis modelassessment of insulin resistance can be used as a screening index ofchanges in insulin sensitivity. HOMA_(IR) can be calculated via standardformula from fasting plasma insulin and glucose as follows:HOMA_(IR)=[Insulin (uU/mL) X glucose (mM)]/22.5.

In some embodiments, insulin signaling can also be measured. Insulinsignaling can be measured by measuring total and phosphorylated Akt,GSK-3β, IGF-1R, IR, IRS-1, p70S6K and PRAS40 in tissue lysates via theLuminex Kits “Akt Pathway Total 7-Plex Panel” (Cat# LHO0002) and “AktPathway Phospho 7-Plex Panel” (Cat# LHO0001) from Invitrogen LifeScience.

The subject application also provides methods of increasingmitochondrial biogenesis in a subject comprising the administration of acomposition disclosed herein to a subject. In various embodiments of theinvention, a composition is administered to the subject in an amountthat delivers synergizing amounts of HMB and resveratrol sufficient toincrease mitochondrial biogenesis within the cells of the subject.Another embodiment provides for the administration of a compositioncomprising synergizing amounts of leucine and resveratrol to the subjectin an amount sufficient to increase mitochondrial biogenesis within thecells of the subject. Yet other embodiments provide for theadministration of a composition comprising synergizing amounts ofleucine, HMB and resveratrol to a subject in an amount sufficient toincrease mitochondrial biogenesis in the subject. Mitochondrialbiogenesis and fat oxidation may be induced in various cells, includingmuscle cells and adipocytes.

Another aspect of the invention provides methods of reducing weight gainor reducing adipose volume in a subject comprising the administration ofcompositions disclosed herein. Body weight can be measured with acalibrated scale and height measured with a wall-mounted stadiometer,and body mass index can be calculated via standard equation (kg/m²). Fatmass can be assessed via dual-energy X-ray absorptiometry at baseline,and 12 and 24 weeks. A LUNAR Prodigy dual-energy X-ray absorptiometrysystem (GE Healthcare, Madison, Wis.), or any other X-ray absorptiometrysystem known in the art, can be maintained and calibrated for use. Aspine phantom can be assessed every day to determine whether any driftin the machine occurred, followed by a daily calibration block.

In this aspect of the invention, a composition is administered to thesubject in an amount that delivers synergizing amounts of HMB andresveratrol sufficient to reduce weight gain in a subject. Anotherembodiment provides for the administration of a composition comprisingsynergizing amounts of leucine and resveratrol to the subject in anamount sufficient to reduce weight gain in the subject. Yet otherembodiments provide for the administration of a composition comprisingsynergizing amounts of leucine, HMB and resveratrol to a subject in anamount sufficient to reduce weight gain in the subject.

Administration of compositions disclosed herein that increase SIRT1 andSIRT3 activity may be useful in any subject in need of metabolicactivation of adipocytes or one or more of their muscles, e.g., skeletalmuscle, smooth muscle or cardiac muscle or muscle cells thereof. Asubject may be a subject having cachexia or muscle wasting. IncreasingSIRT3 activity may also be used to increase or maintain bodytemperature, e.g., in hypothermic subjects and increasing SIRT1 activityis beneficial for treating diabetes (type 2 diabetes) and impairedglucose tolerance and reducing inflammatory responses in a subject.

Increasing SIRT3 activity may also be used for treating or preventingcardiovascular diseases, reducing blood pressure by vasodilation,increasing cardiovascular health, and increasing the contractilefunction of vascular tissues, e.g., blood vessels and arteries (e.g., byaffecting smooth muscles). Generally, activation of SIRT3 may be used tostimulate the metabolism of adipocytes or any type of muscle, e.g.,muscles of the gut or digestive system, or the urinary tract, andthereby may be used to control gut motility, e.g., constipation, andincontinence. SIRT3 activation may also be useful in erectiledysfunction. It may also be used to stimulate sperm motility, e.g., andbe used as a fertility drug. Other embodiments in which it would beuseful to increase SIRT3 include repair of muscle, such as after asurgery or an accident, increase of muscle mass; and increase ofathletic performance.

Thus the invention provides methods in which beneficial effects areproduced by contacting one or more muscle cells with an agent thatincreases the protein or activity level of SIRT3 in the cell. Thesemethods effectively facilitate, increase or stimulate one or more of thefollowing: mimic the benefits of calorie restriction or exercise in themuscle cell, increase mitochondrial biogenesis or metabolism, increasemitochondrial activity and/or endurance in the muscle cell, sensitizethe muscle cell to glucose uptake, increase fatty acid oxidation in themuscle cell, decrease reactive oxygen species (ROS) in the muscle cell,increase PGC-1α and/or UCP3 and/or GLUT4 expression in the muscle cell,and activate AMP activated protein kinase (AMPK) in the muscle cell.Various types of muscle cells can be contacted in accordance with theinvention. In some embodiments, the muscle cell is a skeletal musclecell. In certain embodiments, the muscle cell is a cell of a slow-twitchmuscle, such as a soleus muscle cell.

Resting metabolic rate (RMR)/Substrate Oxidation is measured by indirectcalorimetry using the open circuit technique between the hours of 6 AMand 10 AM after a 12-hour fast and 48-hour abstention from exerciseutilizing a SensorMedics Vmax 29n metabolic cart (Sensor Medics,Anaheim, Calif.). Following a urinary void, the participant restsquietly for 30 minutes in an isolated room with temperature controlled(21-24° C.) environment. The subject is then placed in a ventilated hoodfor a minimum of 30 minutes, until steady state is achieved. Criteriafor a valid measurement can be a minimum of 15 minutes of steady state,with steady state determined as less than 10% fluctuation in minuteventilation and oxygen consumption and less than 5% fluctuation inrespiratory quotient. Metabolic rate is calculated using the Weirequation, RQ is calculated as CO₂ production/O₂ consumption, andsubstrate oxidation is calculated from RQ after correction for urinarynitrogen losses.

Glucose uptake can be measured using in vivo or in vitro techniques. Forexample, glucose uptake can be measured in vivo using a PET scan inconjunction with labeled glucose or glucose analog. Measurements ofglucose uptake can be quantified from the PET scan or by any othertechnique known in the art. In some embodiments, the glucose uptake canbe measured by quantitation of exogenously administered18-F-deoxyglucose uptake via PET.

ROS/Oxidative Stress can be measured by drawing blood into EDTA-treatedtubes, centrifuging to separate plasma, and aliquoting samples forindividual assays. Plasma can be maintained at −80° C. under nitrogen toprevent oxidative changes prior to measurements. Plasma malonaldehyde(MDA) can be measured using a fluorometric assay, and plasma8-isoprostane F_(2α) was measured by ELISA (Assay Designs, Ann Arbor,Mich.).

Another embodiment provides for the administration of a compositioncomprising synergizing amounts of leucine and resveratrol to the subjectin an amount sufficient to increase fatty acid oxidation within thecells of the subject. Yet other embodiments provide for theadministration of a composition comprising synergizing amounts ofleucine, HMB and resveratrol to a subject in an amount sufficient toincrease fatty acid oxidation in the subject.

The compositions can be administered to a subject orally or by any othermethods. Methods of oral administration include administering thecomposition as a liquid, a solid, or a semi-solid that can be taken inthe form of a dietary supplement or a food stuff.

The compositions can be administered periodically. For example, thecompositions can be administered one, two, three, four times a day, oreven more frequent. The subject can be administered every 1, 2, 3, 4, 5,6 or 7 days. In some embodiments, the compositions are administeredthree times daily. The administration can be concurrent with meal timeof a subject. The period of treatment or diet supplementation can be forabout 1, 2, 3, 4, 5, 6, 7, 8, or 9 days, 2 weeks, 1-11 months, or 1year, 2 years, 5 years or even longer. In some embodiments of theinvention, the dosages that are administered to a subject can change orremain constant over the period of treatment. For example, the dailydosing amounts can increase or decrease over the period ofadministration.

The length of the period of administration and/or the dosing amounts canbe determined by a physician, a nutritionist, or any other type ofclinician. The physician, nutritionist, or clinician can observe thesubject's response to the administered compositions and adjust thedosing based on the subject's performance. For example, dosing forsubjects that show reduced effects in energy regulation can be increasedto achieve desired results.

In some embodiments, the compositions administered to a subject can beoptimized for a given subject. For example, the ratio of branched chainamino acids to a sirtuin pathway activator or the particular componentsin a combination composition can be adjusted. The ratio and/orparticular components can be selected after evaluation of the subjectafter being administered one or more compositions with varying ratios ofbranched chain amino acids to a sirtuin pathway activator or varyingcombination composition components.

Another aspect of the invention provides for achieving desired effectsin one or more subjects after administration of a combinationcomposition described herein for a specified time period.

After a period of 6 weeks of administration of the composition, acombination composition comprising (a) a dosing level of resveratrol anda dosing level of HMB or (b) a dosing level of resveratrol and a dosinglevel of leucine can reduce weight gain in the one or more subjects byat least about 10, 15, 20, or 20.5%. The p-value can be less than 0.05(e.g. less than about 0.05, 0.03, 0.02, 0.01, 0.001, 0.0001, or lower).The one or more subjects treated with the same dosing level of one ofthe components (resveratrol, leucine, or HMB) may have insignificantweight reduction, or a weight reduction that is less than about 0, 5, or10%.

After a period of 2 weeks of administration, a composition comprising(a) a dosing level of resveratrol and a dosing level of HMB or (b) adosing level of resveratrol and a dosing level of leucine can increasewhole body fat oxidation in the one or more subjects by at least about10, 15, or 20%. The p-value can be less than 0.05 (e.g. less than about0.05, 0.03, 0.02, 0.01, 0.001, 0.0001, or lower). The increase in wholebody fat oxidation can be sustained while the subjects are administeredthe composition, or for a period of at least 2, 4, 6, 10, 13, 26, or 52weeks. The one or more subjects treated with the same dosing level ofone of the components (resveratrol, leucine, or HMB) may haveinsignificant increase in whole body fat oxidation, or an increase inwhole body fat oxidation that is less than about 0, 5, or 10%.

After a period of 2 weeks of administration, a composition comprising(a) a dosing level of resveratrol and a dosing level of HMB or (b) adosing level of resveratrol and a dosing level of leucine can increasethe thermic effect of food in the one or more subjects by at least about10, 15, 17, or 20%. The p-value can be less than 0.05 (e.g. less thanabout 0.05, 0.03, 0.02, 0.01, 0.001, 0.0001, or lower). The increase inthe thermic effect of food can be sustained while the subjects areadministered the composition, or for a period of at least 2, 4, 6, 10,13, 26, or 52 weeks. The one or more subjects treated with the samedosing level of one of the components (resveratrol, leucine, or HMB) mayhave insignificant increase the thermic effect of food, or an increasethe thermic effect of food that is less than about 0, 5, or 10%.

After a period of 2 weeks of administration, a composition comprising(a) a dosing level of resveratrol and a dosing level of HMB or (b) adosing level of resveratrol and a dosing level of leucine can increasetotal energy expenditure in the one or more subjects by at least about10, 15, 17, or 20%. The p-value can be less than 0.05 (e.g. less thanabout 0.05, 0.03, 0.02, 0.01, 0.001, 0.0001, or lower). The increasetotal energy expenditure can be sustained while the subjects areadministered the composition, or for a period of at least 2, 4, 6, 10,13, 26, or 52 weeks. The one or more subjects treated with the samedosing level of one of the components (resveratrol, leucine, or HMB) mayhave insignificant increase total energy expenditure, or an increasetotal energy expenditure that is less than about 0, 5, or 10%.

The administration of a composition described herein, such as acombination composition, to a subject can allow for the regulation ormaintenance of the subject's energy metabolism. The regulation ormaintenance of energy metabolism can allow for a subject to experience anumber of beneficial effects. These beneficial effects include areduction in weight, a reduction in adipose tissue, an increase in fattyacid oxidation, an increase in browning of adipose tissue (as indiciatedby one or more indicia of fat cell browning), an increase in insulinsensitivity, a decrease in oxidative stress, and/or a decrease ininflammation. Compared to a baseline prior to treatment, these effectscan result in an improvement of about or greater than about 5%, 10%,15%, 20%, 30%, 40%, 50%, 75%, or more. In some embodiments, compared toa baseline prior to treatment, these effects can result in animprovement of about or greater than about 100%, 125%, 150%, 200%, 250%,300%, 400%, 500%, or more. Alternatively, administration of acomposition described herein can allow for maintenance of the subject'sweight, amount of adipose tissue, amount of fatty acid oxidation, levelof insulin sensitivity, oxidative stress level, and/or level ofinflammation. These amounts and/or levels can be maintained within about0%, 1%, 5%, or 10% of the amounts and/or levels at the initiation ofadministration.

The invention provides for a method of treating subjects, comprisingidentifying a pool of subjects amenable to treatment. The identifyingstep can include one or more screening tests or assays. For example,subjects that are identified as diabetic or that have above average orsignificantly greater than average body mass indices and/or weight canbe selected for treatment. The identifying step can include a genetictest that identifies one or more genetic variants that suggest that thesubject is amenable to treatment. The identified subjects can then betreated with one or more compositions described herein. For example,they may be treated with a combination composition comprising a sirtuinpathway activator and a branched-chain amino acid.

The invention also provides for methods of manufacturing thecompositions described herein. In some embodiments, the manufacture of acomposition described herein comprises mixing or combining two or morecomponents. These components can include a sirtuin or AMPK pathwayactivator (such as a polyphenol or polyphenol precursor likeresveratrol, chlorogenic acid, caffeic acid, cinnamic acid, ferulicacid, EGCG, piceatannol, or grape seed extract, or another agent likequinic acid, fucoxanthin, or a PDE inhibitor), branched chain aminoacids or metabolites thereof (such as leucine, valine, isoleucine, HMB,or KIC), and/or an anti-diabetic (such as metformin). In someembodiments, the sirtuin activator is a polyphenol. In otherembodiments, the sirtuin activator is a polyphenol precursor. The amountor ratio of components can be that as described herein. For example, themass ratio of leucine combined with resveratrol can be greater thanabout 80.

In some embodiments, the compositions can be combined or mixed with apharmaceutically active agent, a carrier, and/or an excipient. Examplesof such components are described herein. The combined compositions canbe formed into a unit dosage as tablets, capsules, gel capsules,slow-release tablets, or the like.

In some embodiments, the composition is prepared such that a solidcomposition containing a substantially homogeneous mixture of the one ormore components is achieved, such that the one or more components aredispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules.

Kits

The invention also provides kits. The kits include one or morecompositions described herein, in suitable packaging, and writtenmaterial that can include instructions for use, discussion of clinicalstudies, listing of side effects, and the like. Such kits may alsoinclude information, such as scientific literature references, packageinsert materials, clinical trial results, and/or summaries of these andthe like, which indicate or establish the activities and/or advantagesof the composition, and/or which describe dosing, administration, sideeffects, drug interactions, or other information useful to the healthcare provider. Such information may be based on the results of variousstudies, for example, studies using experimental animals involving invivo models and studies based on human clinical trials. The kit mayfurther contain another agent. In some embodiments, the compound of thepresent invention and the agent are provided as separate compositions inseparate containers within the kit. In some embodiments, the compound ofthe present invention and the agent are provided as a single compositionwithin a container in the kit. Suitable packaging and additionalarticles for use (e.g., measuring cup for liquid preparations, foilwrapping to minimize exposure to air, and the like) are known in the artand may be included in the kit. Kits described herein can be provided,marketed and/or promoted to health providers, including physicians,nurses, pharmacists, formulary officials, and the like. Kits may also,in some embodiments, be marketed directly to the consumer.

EXAMPLES Example 1 Effects of Leucine, KIC and HMB Alone, or inCombination with Resveratrol on Mitochondrial Biogenesis

The experiment shows that Leucine stimulates muscle protein synthesisvia a partially mTOR-independent mechanism. We have shown that catabolicsystems are also stimulated to fuel this process, resulting in increasedmitochondrial biogenesis and fatty acid oxidation (FAO). To address themechanism of this effect, we first determined its mTOR-dependence byassessing leucine stimulation of FAO in the presence and absence of 20nM rapamycin; although rapamycin inhibited FAO in c2c12 myotubes, thedegree of leucine stimulation was preserved (˜50%, p<0.03; FIG. 1).

We next investigated the role of intact leucine (0-0.5 mM) vs. itsmetabolites, α-ketoisocaproic acid (KIC)(0-0.5 mM) and HMB (0-50 μM).All three compounds induced comparable increases in FAO (˜60-70%,p<0.001; FIG. 2). Both leucine and HMB increased myotube mitochondrialbiogenesis (assessed fluorometrically via NAO binding) by ˜50%,(p<0.005, FIG. 3). Consistent with this, HMB and leucine both stimulatedexpression of mitochondrial regulatory (PGC-1α and NRF-1) and component(UCP3) genes (p<0.01, FIG. 4). These data demonstrate that leucinestimulates mitochondrial biogenesis and fatty acid oxidationindependently of mTOR and that these effects appear to be mediated byits metabolite, e.g., HMB.

Example 2 Stimulation of SIRT1 and SIRT 3

The effects of leucine, KIC and HMB on activation of SIRT1 were assessedin the presence and absence of resveratrol in a cell-free system. SIRT1activity was measured by using the SIRT1 Fluorimetric Drug Discovery Kit(BML-AK555, ENZO Life Sciences International, Inc. PA, USA). In thisassay, SIRT1 activity is assessed by the degree of deacetylation of astandardized substrate containing an acetylated lysine side chain. Thesubstrate utilized is a peptide containing amino acids 379-382 of humanp53 (Arg-His-Lys-Lys[Ac]), an established target of SIRT1 activity;SIRT1 activity is directly proportional to the degree of deacetylationof Lys-382. Samples were incubated with peptide substrate (25 μM), andNAD⁺ (500 μM) in a phosphate-buffered saline solution at 37° C. on ahorizontal shaker for 45 minutes. The reaction was stopped with theaddition of 2 mM nicotinamide and a developing solution that binds tothe deacetylated lysine to form a fluorophore. Following 10 minutesincubation at 37° C., fluorescence was read in a plate-readingfluorometer at an excitation wavelength of 360 nm and an emissionwavelength of 450 nm. Resveratrol (100 mM) served as a SIRT1 activator(positive control) and suramin sodium (25 mM) as a SIRT1 inhibitor(negative control). A standard curve was constructed using deacetylatedsubstrate (0-10 μM). Leucine, KIC and HMB all significantly increasedSIRT1 activity in a dose-responsive manner, while valine (a branchedchain amino acid control) exerted no significant effect. FIG. 5demonstrates the effects of leucine and its' metabolites on SIRT1activity at the physiological concentrations of each compound foundafter a leucine-rich meal. As shown in the figure, these effects arequantitatively comparable (and not significantly different from) tothose exerted by a low dose of resveratrol (e.g. 10 μM).

For assaying SIRT3 activity, adipocytes (3T3-L1) were grown toconfluence, differentiated and incubated with leucine (0.5 mM), HMB (5uM), resveratrol (200 nM), HMB (5 uM)+resveratrol (200 nM) or vehiclefor 4 hours. Mitochondrial protein was then isolated from the cells, andSirt3 activity was assessed by fluorometric measurement of deacetylationof a Sirt3 substrate, similar to the methodology described above forSirt1. The Sirt3 substrate was a peptide containing amino acids 317-320of human p53 (Gln-Pro-Lys-Lys[Ac]). Resveratrol, leucine and HMB exertedno significant independent effect on Sirt3 activity. However, combiningresveratrol (200 nM) with HMB (5 uM) resulted in a 58% increase in Sirt3activity (p<0.03, FIG. 6).

Example 3 Leucine and HMB Synergize with Resveratrol to Stimulate FattyAcid Oxidation

Adipocytes (3T3-L1) were grown to confluence, differentiated andincubated with leucine (0.5 mM), HMB (5 uM), resveratrol (200 nM), HMB(5 uM)+resveratrol (200 nM) or vehicle for 4 hours in the presence oflow (5 mM) or high (25 mM) glucose and fatty acid oxidation was measuredusing ³H-palmitate. In the presence of low glucose, only the combinationtreatments (200 nM resveratrol+5 uM HMB; 200 nM resveratrol+0.5 mMleucine) stimulated modest increases in fatty acid oxidation (18%,p<0.05), while the individual components exerted no independent effect(FIG. 7). High glucose medium reduced fatty acid oxidation by 46%(p<0.05). The low dose of resveratrol used exerted no effect on fattyacid oxidation under high glucose conditions, while the leucine and HMBexerted modest, but significant, effects (27% and 29%, respectively,p<0.05 vs. control, FIG. 8). In contrast, the leucine-resveratrol andHMB-resveratrol combinations each exerted a markedly greater effect(118% and 91% stimulation, respectively; p<0.005 vs. control and vs. theindependent effects of leucine, HMB and resveratrol; FIG. 8). These datademonstrate synergy between resveratrol and leucine or its metabolite,HMB, in stimulation of fat oxidation and promotion of a more oxidativephenotype under conditions that model hyperglycemia.

Fatty acid oxidation was measured using ³H-labeled palmitate oxidation,with the ³H label trapped as water as a result of the fat oxidation. The³H was then measured via scintillation counter.

Example 4 Weight Gain, Fat Oxidation, Insulin Sensitivity, andInflammatory Stress in Animals Treated with Resveratrol and Leucine orHMB

Six week old male c57/BL6 mice were fed a high-fat diet with fatincreased to 45% of energy (Research Diets D12451) for 6 weeks to induceobesity. At the end of this obesity induction period, animals wererandomly divided into the following seven different diet treatmentgroups with 10 animals per group (overall 70 animals) and maintained onthese diets for 6 weeks:

-   -   Group 1 (labeled “control group”): high-fat diet only (same as        in obesity induction period (Research Diets D12451)).

This diet was modified for groups 2 to 7 in the following way:

-   -   Group 2 (labeled “low dose resveratrol”): high-fat diet mixed        with 12.5 mg resveratrol/kg diet.    -   Group 3 (labeled “high dose resveratrol”): high-fat diet mixed        with 225 mg resveratrol/kg diet.    -   Group 4 (labeled “low dose HMB”): high-fat diet mixed with 2 g        of the calcium salt of hydroxymethylbutyrate, a naturally        occurring metabolite of leucine (CaHMB).    -   Group 5 (labeled “low dose resveratrol plus low dose CaHMB”):        high fat-diet mixed with 12.5 mg of resveratrol/kg diet and 2 g        CaHMB/kg diet.    -   Group 6 (labeled “low dose resveratrol plus high dose HMB”):        high fat-diet mixed with 12.5 mg of resveratrol/kg diet and 10 g        CaHMB/kg diet.    -   Group 7 (labeled “low dose resveratrol plus leucine”): high        fat-diet mixed with 12.5 mg of resveratrol/kg diet and leucine        increased to 200% of its normal level (from 1.21 to 2.42% by        weight) of the control diet

The animals were housed in polypropylene cages at a room temperature of22±2° C. and regime of 12 h light/dark cycle. The animals had freeaccess to water and their experimental food throughout the experiment.At the of the treatment period (6 weeks) all animals were humanelyeuthanized, and blood and tissues collected for further experiments.

Oxygen consumption/substrate utilization: at the end of the obesityinduction period (day 0 of treatment group) and at 2 weeks and 6 weeksof treatment, oxygen consumption and substrate utilization was measuredvia metabolic chambers using the Comprehensive Lab Animal MonitoringSystems (CLAMS, Columbus Instruments, Columbus, Ohio) in subgroups ofeach treatment group. Each animal was placed in individual cages withoutbedding that allow automated, non-invasive data collection. Each cage isan indirect open circuit calorimeter that provides measurement of oxygenconsumption, carbon dioxide production, and concurrent measurement offood intake. All mice were acclimatized to the chambers for 24 hoursprior to the experiment and maintained under the regular 12:12light:dark cycle with free access to water and food. All experimentswere started in the morning and data were collected for 24 hours. Eachchamber was passed with 0.6 l of air/min and was sampled for 2 min at32-minute intervals. Exhaust O₂ and CO₂ content from each chamber wascompared with ambient O₂ and CO₂ content. Food consumption was measuredby electronic scales.

microPET/CT (glucose and palmitate uptake): at the end of the treatmentperiod (6 weeks of treatment) subgroups of each treatment diet group (5animals/group, 35 animals total) were used to measure whole body glucoseand palmitate uptake via PET/CT Imaging. To visualize these compoundsusing microPET imaging, the glucose or palmitate was labeled withfluorine-18 (108 mins half life) or carbon-11 (20 mins half life),respectively. Each mouse was fasted for 4 hours, then anesthetized using1-3% isoflurane delivered by nose cone or in a mouse-sized inductionchamber purpose-built for small animal imaging protocols. While underanesthesia the mice were injected iv with <2 mCi of each tracer, then beleft for a period of time (minutes to up ˜1 hour) to allow the uptake ofthe tracer. During the scan, mice were kept warm using athermostatically controlled heated bed and were treated with ophthalmicointment prior to scanning Following the live scan the mice werereturned to their cage and revived. Mice were monitored constantlyduring this time. Following live data acquisition the mice weresacrificed by isoflurane overdose and organs harvested for furtherexperiments.

RNA extraction: The Ambion ToTALLY RNA isolation kit (Ambion, Inc.,Austin, Tex., USA) was used to extract total RNA from tissue accordingto the manufacturer's instruction. The concentration, purity and qualityof the isolated RNA will be assessed by measuring the 260/280 ratio(1.8-2.0) and 260/230 ratio (close to 2.0) by using the ND-1000Spectrophotometer (NanoDrop Technologies Inc., Del. USA). Biomarkers ofthe sirtuin-pathway, cytokines, and inflammatory markers (including butnot limited to C-reactive protein, IL-6, MCP-1, and adiponectinmolecules) can be assessed at the RNA level.

Gene Expression: Expression of 18S, Sirt1, Sirt3, PGC1-α, cytochrome coxidase subunit VIIc1 (COX 7), mitochondrial NADH dehydrogenase, nuclearrespiratory factor 1 (NRF1), uncoupling protein (UCP2 (adipocyte)/UCP3(myocyte), p53, AMPK, Akt/PKB, and GLUT4 is measured via quantitativereal-time PCR using an ABI 7300 Real-Time PCR system (AppliedBiosystems, Branchburg, N.J.) with a TaqMan® core reagent kit. Allprimers and probe sets can be obtained from Applied Biosystems TagMan®Assays-on-Demand and utilized accordingly to manufacturer'sinstructions. Pooled RNA from each cell type are serial-diluted in therange of 0.0156-50 ng and were used to establish a standard curve; totalRNA for each unknown sample is also diluted in this range. RT-PCRreactions are performed according to the instructions of the ABIReal-Time PCR system and TaqMan Real Time PCR Core Kit. Expression ofeach gene of interest is then normalized using the corresponding 18Squantitation.

SIRT1 Activity: SIRT1 activity was measured by using the SIRT1Fluorimetric Drug Discovery Kit (BML-AK555, ENZO Life SciencesInternational, Inc. PA, USA). In this assay, SIRT1 activity is assessedby the degree of deacetylation of a standardized substrate containing anacetylated lysine side chain. The substrate utilized is a peptidecontaining amino acids 379-382 of human p53 (Arg-His-Lys-Lys[Ac]), anestablished target of SIRT1 activity; SIRT1 activity is directlyproportional to the degree of deacetylation of Lys-382. Samples wereincubated with peptide substrate (25 μM), and NAD⁺ (500 μM) in aphosphate-buffered saline solution at 37° C. on a horizontal shaker for45 minutes. The reaction was stopped with the addition of 2 mMnicotinamide and a developing solution that binds to the deacetylatedlysine to form a fluorophore. Following 10 minutes incubation at 37° C.,fluorescence was read in a plate-reading fluorometer at an excitationwavelength of 360 nm and an emission wavelength of 450 nm. Resveratrol(100 mM) served as a SIRT1 activator and suramin sodium (25 mM) as aSIRT1 inhibitor; wells including each were utilized as positive andnegative controls in each set of reactions. A standard curve wasconstructed using deacetylated substrate (0-10 μM). Data was normalizedto cellular protein concentration measured via BCA-assay.

Western blot analysis: Tissue samples (adipose and muscle) ishomogenized in ice-cold RIPA lysis buffer containing 150 mM sodiumchloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and 50mM Tris (pH 8.0), aprotinin (1 μg/ml), Leupeptin (10 μg/ml), Pepstatin A(1 μg/ml), 1 mM PMSF, 5 mM EDTA, 1 mM EGTA, 10 mM NaF, 1 mM NaOrthovanadate with an electric homogenizer, then maintained on constantagitation for 2 hours at 4° C. and centrifuged at 4,000 g for 30 min at4° C. Aliquots of supernatants (containing 15-25 of total protein) istreated with 2× Laemmli sample buffer containing 100 mM dithiothreitoland run on 10% (for or 15% SDS-PAGE (for Sirt3). The resolved proteinsis transferred to PVDF membrane and blocked in 5% nonfat dry milk inTris-buffered saline containing 0.1% Tween 10, pH 7.5. After membranesare blocked, the membranes are rinsed in TBST, incubated overnight withappropriate antibody, rinsed in TBST, and incubated for 120 min withhorseradish peroxidase-conjugated anti-rabbit IgG. Antibody-boundprotein is visualized with enhanced chemiluminescence (ECL, Amersham).

The following antibodies are used: Anti-Sirt3 antibody (Cell SignalingTechnology, Beverly, Mass.), Anti-Idh2 (Isocitrate dehydrogenase 2)(Santa Cruz, Calif.), Anti-COX antibody (Santa Cruz).

Low doses of resveratrol and HMB exerted no significant independenteffect on body weight, weight gain, visceral adipose tissue mass, fatoxidation, respiratory exchange ratio (RER), or heat production, whilethe high dose of resveratrol significantly increased both heatproduction and skeletal muscle fat oxidation and decreased RER,indicating a whole-body shift towards fat oxidation (table 1); however,high dose resveratrol exerted no significant effect on body weight,weight gain, or visceral adipose tissue mass. In contrast with the lackof independent effects of a low dose of resveratrol or HMB, combining alow dose of resveratrol with either HMB or leucine resulted insignificant reductions in body weight, weight gain, visceral adiposetissue mass, fat oxidation and heat production, and an associateddecrease in RER (table 1).

TABLE 1 Effects of resveratrol, leucine and HMB on body weight, weightgain, adiposity and fat oxidation in diet-induced obese mice.¹ Low LowResv/ Resv/ Low Low High Low Low High Resv/ Control Resveratrol²Resveratrol³ HMB⁴ HMB HMB⁵ Leucine⁶ P value Weight (g)  40.5 ± 0.5^(a) 40.8 ± 2.5^(a)  38.7 ± 1.2^(a)  40.3 ± 2.1^(a)  36.2 ± 3.2^(b) 34.4 ±1.1^(b)  38.3 ± 2.3^(b) P < 0.05 Weight  22.4 ± 1.1^(a)  20.9 ± 1.5^(a) 22.3 ± 2.4^(a)  22.5 ± 1.2  18.2 ± 1.2^(b) 19.2 ± 1.0^(b)  19.2 ±1.6^(b) p < 0.01 gain (g) Visceral  6556 ± 143  6551 ± 575^(a)  6031 ±323^(a)  6184 ± 460^(a)  5302 ± 324^(b) 4879 ± 243^(b)  4259 ± 321^(b) p< 0.01 Adipose Volume (mm³) Fat  1.34 ± 0.15^(a)  1.51 ± 0.44^(a)  2.29± 0.11^(b)  1.90 ± 0.29^(a)  2.09 ± 0.30^(b)  1.97 ± 0.28^(b)  1.76 ±0.09^(a,b) P < 0.05 oxidation (PET palmitate uptake; Muscle SUV)Respiratory 0.850 ± 0.008^(a) 0.847 ± 0.008^(a) 0.825 ± 0.007^(b) 0.844± 0.012^(a) 0.815 ± 007^(b) 0.8818 ± 0.09^(b)  0.811 ± 0.010^(b) P <0.01 Exchange Ratio (24 hr RER) Heat 0.521 ± 0.015^(a) 0.517 ± 0.014^(a)0.552 ± 0.015^(b) 0.526 ± 0.011^(a) 0.544 ± 0.010^(b)  0.547 ± 0.009^(b)0.550 ± 0.012^(b) P < 0.05 Production ¹non-matching letter superscriptsin each row denote significant differences at the indicated p value ²Lowresveratrol: 12.5 mg resveratrol/kg diet ³High resveratrol: 225 mgresveratrol/kg diet ⁴Low HMB: 2 g hydroxymethylbutyrate (calcium salt)⁵Leucine: Leucine increased two-fold, from 1.21% in other diets to 2.42%

Table 2 shows the effects of the dietary treatments on indices ofinsulin sensitivity. None of the treatments exerted any effect on plasmaglucose. Neither resveratrol at either dose nor HMB exerted anysignificant effect on plasma insulin or on muscle glucose uptake.However, the combination of a low dose of resveratrol with either HMB orleucine resulted in significant, marked decreases in plasma insulin.This reduction in insulin with no change in plasma glucose reflectssignificant improvements in muscle and whole-body insulin sensitivity,as demonstrated by significant and substantial decreases in HOMA_(IR)(homeostatic assessment of insulin resistance) and correspondingincreases in skeletal muscle ¹⁸F-deoxyglucose uptake (table 2 and FIG.9).

TABLE 2 Effects of resveratrol, leucine and HMB on indices of insulinsensitivity in diet- induced obese mice.¹ Low Low Resv/ Resv/ Low LowHigh Low Low High Resv/ Control Resveratrol² Resveratrol³ HMB⁴ HMB HMB⁵Leucine⁶ P value Glucose 4.97 ± 0.60^(a) 5.14 ± 0.85^(a) 5.14 ± 0.75^(a)4.28 ± 0.49^(a) 4.67 ± 0.49^(a) 4.33 ± 0.41^(a) 5.05 ± 0.92^(a) NS (mM)Insulin 12.5 ± 3.4^(a) 10.4 ± 1.6^(a) 10.1 ± 2.7^(a)  8.3 ± 1.1^(a)  5.8± 0.7^(a)  3.9 ± 1.2^(b)  5.5 ± 1.4^(a) P < 0.005 (μU/mL) HOMA_(IR) 2.61± 0.82^(a) 2.41 ± 0.66^(a) 0.59 ± 0.26^(b) 1.93 ± 0.32^(a) 1.18 ±0.25^(c) 0.87 ± 0.31^(b) 1.14 ± 0.37^(c) P < 0.01 Muscle 3.64 ± 0.88^(a)3.63 ± 1.29^(a) 3.87 ± 0.32^(a) 2.99 ± 0.42^(a) 5.90 ± 0.41^(b) 5.93 ±1.63^(b) 5.68 ± 0.75^(b) P < 0.02 Glucose Uptake (¹⁸F- deoxyglucose SUV)¹non-matching letter superscripts in each row denote significantdifferences at the indicated p value ²Low resveratrol: 12.5 mgresveratrol/kg diet ³High resveratrol: 225 mg resveratrol/kg diet ⁴LowHMB: 2 g hydroxymethylbutyrate (calcium salt) ⁵Leucine: Leucineincreased two-fold, from 1.21% in other diets to 2.42%

FIG. 10 shows the effects of dietary treatments on adipose tissue Sirt1activity. Neither resveratrol nor HMB exerted significant independenteffects on Sirt1 activity, although high dose resveratrol exhibited anon-significant trend towards an increase. In contrast, combining a lowdose of resveratrol with either HMB or leucine resulted in ˜two-foldincreases in tissue Sirt1 activity. Such sirtuin activation would beanticipated to reduce inflammatory response. Consistent with thisconcept, the high dose of resveratrol significantly reduced circulatingIL-6, while the combination of a low dose of resveratrol (which exertedno independent effect) with HMB resulted in a markedly greater loweringof IL-6 (table 3). Similarly, while neither HMB nor a low dose ofresveratrol exerted any effect on MCP-1 or c-reactive protein, thecombination of a low dose of resveratrol with either HMB or leucineresulted in significant decreases in both inflammatory biomarkers.Moreover, the anti-inflammatory cytokine adiponectin was increased inresponse to a low dose of resveratrol in combination with either HMB orleucine, while the individual components at these doses exerted nosignificant effect (table 3).

TABLE 3 Effects of resveratrol, leucine and HMB on inflammatorybiomarkers in diet- induced obese mice.¹ Low Low Resv/ Resv/ Low LowHigh Low Low High Resv/ Control Resveratrol² Resveratrol³ HMB⁴ HMB HMB⁵Leucine⁶ P value C- 95.6 ± 9.6^(a) 134.8 ± 8.5^(a)  123.9 ± 35.3^(a)98.6 ± 5.1   67.4 ± 12.2^(b)  58.3 ± 12.4^(b)  55.9 ± 17.7^(b) P < 0.01reactive (ng/mL) IL-6 29.0 ± 6.4^(a) 23.2 ± 2.9^(a) 14.1 ± 1.3^(b) 19.9± 3.1^(a)  6.9 ± 1.2^(c)  4.5 ± 2.6^(c) 11.2 ± 4.1^(b) P < 0.005 (pg/mL)MCP-1 115.8 ± 19.7^(a) 104.4 ± 16.5^(a) 27.3 ± 6.8^(b) 116.8 ± 9.3^(a) 24.2 ± 6.2^(b) 15.2 ± 3.7^(b) 34.9 ± 5.9^(b) P < 0.001 (pg/mL) Adipo-11.0 ± 0.9^(a) 12.4 ± 1.1  14.8 ± 1.8^(b) 11.1 ± 1.6^(a) 14.1 ± 0.8^(b)16.3 ± 3.0^(b) 14.5 ± 1.0^(b) P < 0.03 nectin (ng/mL) ¹non-matchingletter superscripts in each row denote significant differences at theindicated p value ²Low resveratrol: 12.5 mg resveratrol/kg diet ³Highresveratrol: 225 mg resveratrol/kg diet ⁴Low HMB: 2 ghydroxymethylbutyrate (calcium salt) ⁵Leucine: Leucine increasedtwo-fold, from 1.21% in other diets to 2.42%

Collectively, these data demonstrate synergy between low doses ofresveratrol and leucine or its metabolite HMB in activating Sirt1 andSirt1-dependent outcomes. These include increased fat oxidation andattenuation of adiposity and obesity, augmentation of insulinsensitivity and reversal of insulin resistance, and attenuation ofsystemic inflammatory stress.

Example 5 Synergistic Effects of Polyphenols and Related Compounds onSirtuin Activation and Downstream Pathways

All compounds were tested for potential to independently orsynergistically modulate sirtuin signaling either by direct stimulationor indirect via upstream signaling via AMPK. A key outcome of Sirt1signaling is stimulation of PGC1-α and subsequent stimulation ofmitochondrial biogenesis and fatty acid oxidation. Accordingly, fattyacid oxidation, measured as palmitate-induced oxygen consumption asdescribed below, was utilized as a sensitive first level of screeningfor aerobic mitochondrial metabolism. A dose-response curve for fattyacid oxidation was established for each compound studied, and a“sub-therapeutic dose” was defined as the highest dose that exerted noeffect in this system. This dose, typically found to be in the 200-1000nM range for most compounds studied, was then used to evaluatesynergistic effects with leucine, HMB, or sub-therapeutic doses of othercompounds. These experiments were conducted in fully differentiatedadipocytes (3T3-L1) and myotubes (C2C12). To evaluate the impact ofthese combinations on cross-talk between adipose and muscle tissues,adipocytes were treated for 48 hours, the medium collected (conditionedmedia, CM) and then exposed to myotubes; similar experiments wereconducted with myotubes treated, CM collected, and exposed toadipocytes. Following assessment of fatty acid oxidation, Sirt1activity, AMPK activity, mitochondrial biogenesis and glucoseutilization (measured as glucose-induced extracellular acidification inthe absence of fatty acids in the media) were assessed for leadcombinations and appropriate controls.

Cell Culture:

C2C12 and 3T3-L1 preadipocytes (American Type Culture Collection) wereplated at a density of 8000 cells/cm² (10 cm² dish) and grown inDulbecco's modified eagle's medium (DMEM) containing 10% fetal bovineserum (FBS), and antibiotics (growth medium) at 37° C. in 5% CO₂.Confluent 3T3-L1 preadipocytes were induced to differentiate with astandard differentiation medium consisting of DMEM medium supplementedwith 10% FBS, 250 nM dexamethasone, 0.5 mM 3-Isobutyl-1-methylxanthine(IBMX) and 1% penicillin-streptomycin. Preadipocytes were maintained inthis differentiation medium for 3 days and subsequently cultured ingrowth medium. Cultures were re-fed every 2-3 days to allow >90% cellsto reach fully differentiation before conducting chemical treatment. Fordifferentiation of C2C12 cells, cells were grown to 100% confluence,transferred to differentiation medium (DMEM with 2% horse serum and 1%penicillin-streptomycin), and fed with fresh differentiation mediumevery day until myotubes were fully formed (3 days).

Measurements:

Fatty Acid Oxidation:

Cellular oxygen consumption was measured using a Seahorse BioscienceXF24 analyzer (Seahorse Bioscience, Billerica, Mass.) in 24-well platesat 37° C., as described by Feige et al. (Feige J, Lagouge M, Canto C,Strehle A, Houten S M, Milne J C, Lambert P D, Mataki C, Elliott P J,Auwerx J. Specific SIRT1 activation mimics low energy levels andprotects against diet-induced metabolic disorders by enhancing fatoxidation. Cell Metabolism 2008; 8:347-358) with slight modifications.Cells were seeded at 40,000 cells per well, differentiated as describedabove, treated for 24 hours with the indicated treatments, washed twicewith non-buffered carbonate-free pH 7.4 low glucose (2.5 mM) DMEMcontaining carnitine (0.5 mM), equilibrated with 550 μL of the samemedia in a non-CO₂ incubator for 45 minutes, and then inserted into theinstrument for 15 minutes of further equilibration, followed by O₂consumption measurement. Three successive baseline measures atfive-minute intervals were taken prior to injection of palmitate (200 μMfinal concentration). Four successive 5-minute measurements of O₂consumption were then conducted, followed by 10 minute re-equilibrationand another 3-4 5-minute measurements. This measurement pattern was thenrepeated over a 4-6 hour period. Data for each sample were normalized tothe pre-palmitate injection baseline for that sample and expressed as %change from that baseline. Pre-palmitate injection values were 371±14pmol O₂/minute for myotubes and 193±11 pmol O₂/minute for adipocytes.The area under of the curve of O₂ consumption change from baseline foreach sample was then calculated and used for subsequent analysis.

Glucose Utilization:

In the absence of a fatty acid source and oxidative metabolism,glycolysis and subsequent lactate production results in extracellularacidification, which was also measured using a Seahorse Bioscience XF24analyzer. Cells were prepared and equilibrated similar to the methodsdescribed above for fatty acid oxidation, with the exclusion ofcarnitine from the medium. Following instrument equilibration and threebaseline measurements, glucose was injected to a final concentration of10 mM in each well. Measurements were taken as described above utilizingthe sensors for extracellular acidification rather than O₂ consumption.Insulin (final concentration of 5 nM) was added to some wells as apositive control. Data for each sample were normalized to thepre-glucose injection baseline for that sample and expressed as % changefrom that baseline. The area under of the curve of extracellularacidification change from baseline for each sample was then calculatedand used for subsequent analysis.

Mitochondrial Biogenesis:

Mitochondrial biogenesis was assessed as change in mitochondrial mass,as described by Sun et al. (Sun X and Zemel MB (2009) Leucine modulationof mitochondrial mass and oxygen consumption in skeletal muscle cellsand adipocytes. Nutrition and Metabolism 6:26(doi:10.1.1186/1743-707S-6-26)). The mitochondrial probe NAO(Invitrogen, Carlsbad, Calif.) was used to analyze mitochondrial mass byfluorescence (excitation 485 nm and emission 520 nm). and quantitativedata were obtained with a fluorescence microplate reader (Synergy HT,BioTek Instruments, Winooski, Vt.). The intensity of fluorescence wasexpressed as arbitrary units per μg protein and normalized to controlvalues within each assay.

AMPK Activity:

AMP-activated protein kinase (AMPK) was measured using a commercial kit(CycLex AMPK Kinase Assay Kit, CycLex Co, Ltd, Nagano, Japan). The assayis based upon AMPK phosphorylation of IRS-1 S789. Phosphorylated IRS-1S789 is then detected by an anti-phospho-mouse IRS-1 S789 monoclonalantibody, which is then bound to horseradish peroxidase conjugatedanti-mouse IgG which catalyzes a chromogenic reaction withtetra-methylbenzidine. Color formation is proportional to AMPK activityand was measured in 96-well ELISA plates at dual wavelengths (450/540nm) using a microplate reader (Synergy HT, BioTek Instruments, Winooski,Vt.). These values are expressed as fluorescent units/mg protein andnormalized to control values within each assay.

Sirt1 Activity:

SIRT1 activity was measured by using the SIRT1 Fluorimetric DrugDiscovery Kit (BML-AK555, ENZO Life Sciences International, Inc. PA,USA). The assay measures SIRT1 activity by the degree of deacetylationof a standardized substrate containing an acetylated lysine side chain.The substrate utilized is a peptide containing amino acids 379-382 ofhuman p53 (Arg-His-Lys-Lys[Ac]), an established target of SIRT1activity; SIRT1 activity is directly proportional to the degree ofdeacetylation of Lys-382. Samples were incubated with peptide substrate(25 μM), and NAD⁺ (500 μM) in a phosphate-buffered saline solution at37° C. on a horizontal shaker for 45 minutes. The reaction was stoppedwith the addition of 2 mM nicotinamide and a developing solution thatbinds to the deacetylated lysine to form a fluorophore. Following 10minutes incubation at 37° C., fluorescence was read in a plate-readingfluorometer (Synergy HT, BioTek Instruments, Winooski, Vt.) at anexcitation wavelength of 360 nm and an emission wavelength of 450 nm.Resveratrol (100 mM) served as a SIRT1 activator (positive control) andsuramin sodium (25 mM) as a SIRT1 inhibitor (negative control). Astandard curve was constructed using deacetylated substrate (0-10 μM).

Statistics:

Data were analyzed via one-way analysis of variance and leastsignificant difference test was used to separate significantly differentgroup means

Results:

Resveratrol-Leucine and Resveratrol-HMB:

Leucine (0.5 mM) and HMB (5 μM) stimulated Sirt1 activity and fatty acidoxidation by 30-50%, similar to the effects of 10 resveratrol, whilelower levels of resveratrol (here 200 nM) exerted no effect; leucine,HMB and a low dose of resveratrol exerted no independent effects onSirt3. However, the combination of either leucine or HMB with 200 nMresveratrol resulted in a ˜90% stimulation of Sirt1, a ˜60% stimulationof both Sirt3 and 91%-118% increases in fatty acid oxidation (p<0.005).

The concentrations of leucine and HMB in all experiments described beloware 0.5 mM (leucine) and 5 μM (HMB). Each of the compounds studied incombination with leucine or HMB were studied at concentrations thatexerted no independent effect on the variables under study in order toassess potential synergies. These concentrations are defined for eachcompound below.

Chlorogenic Acid:

Chlorogenic acid is a naturally occurring polyphenol described as ahydroxycinnamic acid; it is an ester of caffeic acid and L-quinic acid(evaluated below). Chlorogenic acid dose-response curves indicateconcentrations of 500 nM or below exert no effect; accordingly, this wasthe concentration used in synergy experiments.

FIG. 12 shows the effects of the chlorogenic acid combinations inmyotubes, and significant quanitative data are summarized in FIG. 3.Chlorogenic acid (500 nM)/HMB elicited a 42% increase in fatty acidoxidation with 6 hour treatment (p=0.003) and 441% over 24 hours(p=0.05) in skeletal muscle cells (myotubes), while no significanteffect was observed in adipocytes. Notably, adding resveratrol (200 nM)attenuated or eliminated these effects, suggesting potential competitionfor a common site of action (FIG. 13).

The chlorogenic acid/HMB combination stimulated adipocyte Sirt1 activity40% (p=0.005) while the chlorogenic acid/leucine combination stimulatedSirt1 by 67% (p=0.0001) (FIG. 14) and more modestly stimulated AMPKactivity (30-35%, NS: p=0.078). In contrast to myotubes, the chlorogenicacid/HMB and chlorogenic acid/leucine combinations exerted no directeffect on adipocyte fatty acid oxidation; however, adipocyte conditionedmedia experiments demonstrated that treatment of adipocytes with thesecombinations for 48 hours resulted in conditioned media that stimulatedmyotube fatty acid oxidation by 76% (p=0.013).

Both chlorogenic acid-leucine and chlorogenic acid-HMB exertedsignificant effects on glucose utilization as measured by extracellularacidification responses to glucose addition (chlorogenic acid-leucine:53%, p=0.007; chlorogenic acid-HMB: 35%, p=0.045; FIG. 15).

Caffeic Acid:

Caffeic acid is another naturally occurring phenolic compound describedas another hydroxycinnamic acid. Caffeic acid dose-response curvesindicate concentrations of 1 μM or below exert no effect; accordingly,this was the concentration used in synergy experiments.

FIGS. 16 and 17 show the effects of the caffeic acid combinations inmyotubes, and the quantitative data is summarized in FIG. 18. Thecaffeic acid-leucine combination exerted a modest, non-statisticallysignificant increase in myotube fatty acid oxidation (35%), while thecaffeic acid-HMB combination exerted significant effects on fatty acidoxidation in both adipocytes (361%, p=0.05) and myotubes (182%,p=0.016). These effects were inhibited by the addition of 200 nMresveratrol, suggesting competition, similar to that seen withchlorogenic acid (FIG. 17).

Quinic Acid:

Quinic acid is a naturally occurring polyol found in coffee beans andsome other plant products. Although not a polyphenol, it is evaluatedhere because it is a component of chlorogenic acid and may be producedvia hydrolysis of chlorogenic acid. Quinic acid dose-response curvesindicate concentrations of 500 nM or below exert no effect; accordingly,this was the concentration used in synergy experiments.

FIGS. 19 and 20 show the effects of the quinic acid combinations inadipocytes, and the quantitative data is summarized in FIG. 21. Quinicacid-HMB and quinic acid-leucine combinations produced robust increasesin adipocyte fatty acid oxidation (141% for the quinic acid-HMBcombination, p=0.05; 320% for the quinic acid-leucine combination,p=0.012; FIG. 21) and more modest increases in myotubes (˜30%, p=0.03).Unlike chlorogenic acid and caffeic acid, addition of resveratrol (200nM) did not attenuate these effects. The quinic acid combinations appearnot to exert their effects directly on Sirt1, as there was no short-termeffect on Sirt1 activity, and instead acts upstream with a significantincrease in AMPK activity (47%, p<0.0001; FIG. 22). Both the quinicacid-leucine and quinic acid-HMB combinations exerted significanteffects on glucose utilization as measured by extracellularacidification responses to glucose addition in both adipocytes andmyotubes (quinic acid-HMB, 99%, p=0.05; quinic acid-leucine, 224%,p=0.0003; FIG. 23).

Other Polyols:

As noted above, quinic acid was evaluated as a hydrolysis product ofchlorogenic acid. To determine if the robust effects of quinic acidreflected the effects of a unique molecule (quinic acid) or polyols as aclass of compounds, other polyols were evaluated, as follows. These datasuggest that effects of quinic acid are not readily extrapolated toother polyols.

Sorbitol is a sugar alcohol analogue of glucose. Sorbitol dose-responsecurves indicate concentrations of 500 nM or below exert no effect;accordingly, this was the concentration used in synergy experiments.Addition of this level of sorbitol to either HMB or leucine resulted instimulation of myotube fatty acid oxidation (44-70%, p=0.023). However,these effects are not significantly different from the independenteffects of leucine and HMB in the absence of sorbitol, indicating nosynergy.

Myo-inositol is a polyol metabolite of glucose. Myo-inositoldose-response curves indicate concentrations of 100 nM or below exert noeffect; accordingly, this was the concentration used in synergyexperiments. Combining 100 nM myo-inositol with leucine or HMB produced60% increase in fat oxidation, comparable to the independent effects ofleucine and HMB in the absence of myo-inositol, indicating no synergy.

Maltitol is a disaccharide made by hydrogenation of maltose. Maltitoldose-response curves indicate concentrations of 100 nM or below exert noeffect; accordingly, this was the concentration used in synergyexperiments. However, no synergy was noted.

Cinnamic Acid:

Cinnamic acid is a naturally occurring phenolic found in cinnamon oil.It bears strong structural homology to both caffeic acid and chlorogenicacid. Cinnamic acid dose-response curves indicate concentrations of 500nM or below exert no effect; accordingly, this was the concentrationused in synergy experiments.

The cinnamic acid combinations exerted robust effects in both adipocytesand myotubes. FIGS. 24 and 25 show the effects of the cinnamic acidcombinations in myotubes, and the quantitative data for adipocytes andmyotubes is summarized in FIGS. 26 and 27, respectively. Cinnamicacid-HMB and cinnamic acid-leucine combinations increased adipocytefatty acid oxidation by 290% (p=0.004) and 1227% (p=0.006), respectively(FIG. 26). In myotubes, the same combinations increased fatty acidoxidation by 199% (p=0.02) and 234% (p=0.05) (FIG. 27). Further,treatment of adipocytes with these cinnamic acid combinations to produceadipocyte conditioned media which was then applied to myotubes resultedin a 273% increase in myotube fatty acid oxidation (p=0.0002). As withquinic acid, these effects were not attenuated by the addition of 200 nMresveratrol and there was no short-term effect on Sirt1 activity.Instead, the primary effect of these combinations appears to beAMPK-mediated, with Sirt1 effects occurring downstream over a longerperiod of time, as the combinations resulted in 136-157% increases inAMPK activity (p=0.0001; FIG. 28).

Ferulic Acid:

Ferulic acid is another hydroxycinnamic acid. Ferulic acid is naturallyoccurring in coffee and apples, as well as some other fruits, legumesand grains. Ferulic acid dose-response curves indicate concentrations of500 nM or below exert no effect; accordingly, this was the concentrationused in synergy experiments. Ferulic acid combinations exerted strongeffects on fatty acid oxidation. The ferulic acid-HMB combinationincreased fatty acid oxidation by 1281% (p=0.018) in adipocytes (FIGS.29 and 30) and by 82% in myotubes (p=0.05) (FIGS. 31 and 32). However,the ferulic acid-leucine combination exerted no significant effect inadipocytes (FIG. 30), but increased fatty acid oxidation by 137% inmyotubes (p=0.034; FIG. 32). Similar to cinnamic acid, the effects ofthe ferulic acid-HMB combination in adipocytes and the ferulicacid-leucine combination in myocytes were not attenuated by the additionof resveratrol and there was no short-term direct effect on Sirt1activity, but there was a significant stimulation of AMPK activity(55-62%, p=0.05; FIG. 33).

Piceatannol:

Piceatannol is a polyphenol classified as a stilbene. It is a metaboliteof resveratrol and is naturally occurring in red wine. Piceatannoldose-response curves indicate concentrations of 1 nM or below exert noeffect; accordingly, this was the concentration used in synergyexperiments. To date, only fatty acid oxidation experiments have beenconducted (FIGS. 34-36). Data from these experiments demonstratesignificant effects of both combinations in both adipocytes andmyotubes. The piceatannol-leucine combination elicited a 73% increase infatty acid oxidation in adipocytes (p=0.05) and a 2301% increase infatty acid oxidation in myotubes (p=0.039), and the piceatannol-HMBcombination elicited a 60% increase in adipocytes (p=0.05) and a 6085%increase in myotubes (FIG. 36).

Ellagic Acid:

Ellagic acid is a large polyphenol naturally occurring in strawberries,raspberries and grapes, as well as a number of other plant products.This polyphenol failed to exert a significant effect in most of ourassays, and dose-response curves of ellagic acid indicated littleactivity, even at high concentrations (50 μM).

Epigallocatechin Gallate (EGCG):

EGCG is a polyphenol ester of epigallocatechin and gallic acid. EGCG isthe predominant catechin in green tea. Despite claims to the contrary,we find this compound to be minimally active in directly stimulatingfatty acid oxidation and do not detect synergistic effects with eitherHMB or leucine in stimulating fatty acid oxidation. However, EGCG (1 μM)did exert significant effects on glucose utilization as measured byextracellular acidification. This level of EGCG exerted no independenteffect on glucose utilization, but stimulated a 94% increase in glucoseutilization when combined with HMB (p=0.015; FIG. 37) and a 156%increase in glucose utilization when combined with leucine (p=0.017;FIG. 37). Notably, adding resveratrol to this combination exerted noadditional effect, but also did not attenuate the observed effects. Theeffects of these combinations on AMPK and Sirt1 activities have not yetbeen determined.

Fucoxanthin:

Fucoxanthin is a non-polyphenolic pigment found in brown seaweed (“SeaMustard”; Undaria pinnatifida). Fucoxanthin dose-response curvesindicate concentrations of 100 nM or below exert no effect; accordingly,this was the concentration used in synergy experiments.

Fucoxanthin-HMB and fucoxanthin-leucine combinations both exerted potenteffects on fatty acid oxidation in adipocytes (fucoxanthin-HMB, 425%increase, p=0.033; fucoxanthin-leucine, 148% increase, p=0.05; FIGS.38-40) and myotubes (fucoxanthin-HMB, 236% increase, p=0.05;fucoxanthin-leucine, 82% increase, p=0.024). Addition of resveratrolneither attenuated nor augmented these effects.

Fucoxanthin combination with both HMB and leucine significantlyaugmented glucose utilization in myotubes and adipocytes (FIGS. 41 and42). The fucoxanthin-HMB combination resulted in a 59% increase(p=0.038) and the fucoxanthin-leucine combination resulted in a 63%increase (p=0.034) in myotubes (FIG. 41). In adipocytes, thefucoxanthin-HMB combination resulted in a 321% increase (p=0.02) and thefucoxanthin-leucine combination resulted in a 557% increase (p=0.003;FIG. 42).

The effects of the fucoxanthin combinations on AMPK and Sirt1 activityhave not yet been determined.

Grape Seed Extract:

Grape seed extract (GSE) is an undifferentiated mixture of polyphenols,including resveratrol, and other naturally occurring compounds in grape.It was selected for study as a broad example of synergy with a naturallyoccurring group of polyphenols. Since it is a mixture, it is notpossible to define concentrations in molar units, so mass units are usedfor this section. GSE dose-response curves indicate concentrations of 1μg/mL or below exert no effect; accordingly, this was the concentrationused in synergy experiments. GSE-leucine increased adipocyte fatty acidoxidation by 74%, but this did not reach statistical significance. TheGSE-HMB combination increased fatty acid oxidation by 2262% (p=0.04;FIGS. 43 and 44). The effects of both combinations were attenuated bythe addition of resveratrol to the combinations (FIG. 44). GSE-leucineand GSE-HMB combinations modestly increased both AMPK activity (40-80%,p<0.01; FIG. 45) and Sirt1 activity (15-20%, p<0.03).

Metformin:

Metformin, a biguanide, is a commonly prescribed oral hypoglycemicagent. Its known mechanism of action is via stimulation of AMPK,resulting in increased insulin sensitivity as well as increased fatoxidation. Thus, metformin, HMB, leucine, and several of the polyphenolsdiscussed above converge on the same signaling pathways. Accordingly, wesought to determine whether combinations of metformin with thesecompounds exert a synergistic effect, thereby lowering the concentrationof metformin necessary to achieve therapeutic effect.

Metformin dose-response curves indicate concentrations of 0.1 mM orbelow exert no effect; accordingly, this was the concentration used insynergy experiments. This level is substantially lower thanconcentrations used to assess independent effects of metformin incellular studies (2-10 mM). Combining metformin with resveratrol (200nM) and HMB resulted in a 1607% increase in myotube fatty acid oxidation(p=0.0001; FIG. 46), while the metformin-leucine-resveratrol combinationelicited a 1039% increase (p=0.001). Omitting resveratrol from thecombinations resulted in statistically significant, but more modest,synergistic interactions with metformin (FIG. 46). Metformin-HMBelicited a 58% increase in myotube fatty acid oxidation (p=0.05) whilemetformin-leucine elicited a 176% increase (p=0.03). These combinationsalso significantly augmented glucose utilization in myotubes by 61 and51%, respectively (p=0.028 for both). Both metformin-HMB andmetformin-leucine stimulated myotube glucose utilization by 50-60%(p=0.03; FIG. 47).

Consistent with these data, these combinations also significantlyincreased AMPK activity (FIG. 48). The metformin-HMB combinationincreased myotube AMPK activity by 50% (p=0.031) and themetformin-leucine combination by 22%. Inclusion of resveratrol (200 nM)significantly augmented these effects; metformin-HMB-resveratrolincreased AMPK activity by 86% (p=0.026) and themetformin-leucine-resveratrol combination resulted in a 95% increase(p=0.017). These combinations exerted similar effects on Sirt1 activity.Metformin-HMB increased Sirt1 activity by 38% and 58% in adipocytes andmyotubes, respectively (p=0.001 for both). Comparable effects wereobserved for mitochondrial biogenesis (metformin-HMB-resveratrol, 35%,p=0.001; metformin-leucine-resveratrol, 27%, p=0.013; FIG. 49).

Notably, combining metformin with either grape seed extract orchlorogenic acid resulted in similar stimulation of Sirt1 activity.Metformin-grape seed extract increased activity by 24% (p=0.001) andmetformin-chlorogenic acid increased activity by 42% (p=0.004).

Rosiglitazone:

Rosiglitazone is an oral hypoglycemic agent in the thiazolidinedione(TZD) class. Its adverse event profile has raised significant concern,limiting its current use, although it is still approved. TZDs act bybinding to peroxisome proliferator-activated receptor gamma (PPARγ). Oneof the targets of PPARγ is peroxisome proliferator-activated receptorgamma coactivator 1-alpha (PGC-1α), a regulator of mitochondrialbiogenesis and fatty acid oxidation that is a downstream mediator ofSirt1. Accordingly, we sought to determine whether combinations ofrosiglitazone with the compounds investigated here exert a synergisticeffect, thereby lowering the concentration of metformin necessary toachieve therapeutic effect.

Rosiglitazone dose-response curves indicate concentrations below 1 nMexert no effect; accordingly, this was the concentration used in synergyexperiments. This level is lower than that typically used in cellculture experiments (10 nM-10 μM) and is markedly lower than plasmalevels typically achieved following IV or oral dosing (400 nM-1.7 μM).

Combining rosiglitazone with either leucine or HMB resulted insignificant stimulation of fatty acid oxidation in both mytubes (FIG.50) and adipocytes (FIG. 51). The rosiglitazone-HMB combinationstimulated fatty acid oxidation by 521% (p=0.004), and therosiglitazone-leucine combination stimulated fatty acid oxidation by231% (p=0.023) and myotube fatty acid oxidation by 92% (p=0.009).Combining rosiglitazone with resveratrol (200 nM) also resulted instimulation of fatty acid oxidation (177%, p=0.003); however, addingresveratrol to the rosiglitazone-HMB or rosiglitazone-leucinecombinations was not more effective than the combinations in the absenceof resveratrol in myotubes and attenuated the effects of thecombinations in adipocytes.

Combining rosiglitazone with either HMB or leucine resulted in markedincreases in glucose utilization (FIG. 52). The rosiglitazone-HMBcombination stimulated a 322% increase (p=0.05) and therosiglitazone-leucine combination stimulated a 341% increase. Acomparable increase was found when resveratrol (200 nM) was combinedwith rosiglitazone (415%, p=0.001), but adding resveratrol to either therosiglitazone-HMB or rosiglitazone-leucine combinations did not furtheraugment glucose utilization.

Phosphodiesterase (PDE) Inhibitors:

The effects of resveratrol on Sirt1 activation may be mediated, in part,via inhibiting cAMP Phosphodiesterase, resulting in upregulation of AMPKand subsequent activation of Sirt1 rather than a direct effect. However,other this effect may only be relevant at high (>50 μM) resveratrolconcentrations. Accordingly we have evaluated the effects of variousnon-specific PDE inhibitors, as follows.

Caffeine is a naturally occurring methyl-xanthine found primarily incoffee, tea, guarana and yerba mate. Caffeine is both an adenosineantagonist and a non-specific PDE inhbitor. Caffeine dose-responsecurves indicate concentrations below 10 nM exert no effect; accordingly,this was the concentration used in synergy experiments. This level is˜0.1% of the plasma concentration observed following caffeineconsumption (1-10 μM). Combining 10 nM of caffeine with resveratrol (200nM) resulted in a 254% increase in fatty acid oxidation in myotubes(p=0.03; FIG. 53), while neither component exerted an independenteffect. Combining caffeine with 0.5 mM leucine stimulated adipocytefatty acid oxidation by 732% (p=0.008; FIGS. 54 and 55), and combiningcaffeine with 5 μM HMB resulted in a 334% increase in fat oxidation inmyotubes (p=0.05; FIG. 53). The caffeine-leucine combination alsomarkedly improved muscle cell glucose utilization as measured byextracellular acidification responses to glucose addition (574%improvement, p=0.003). Caffeine also exhibited significant synergy withmetformin (0.1 mM), resulting in a 240% increase in myotube fatty acidoxidation (p=0.013; FIG. 53), although it did not exert a synergisticeffect on glucose utilization.

Theophylline is a metabolite of caffeine that is also naturallyoccurring in tea and cocoa. Theophylline dose-response curves indicateconcentrations below 1 μM exert no effect; accordingly, this was theconcentration used in synergy experiments. Combining theophylline with 5μM HMB resulted in a 396% increase in myotube fatty acid oxidation(p=0.03; FIG. 56). Similar synergy occurred between theophylline andresveratrol (486%, p=0.03), while combining HMB, resveratrol and HMB didnot further augment this effect (382%, p=0.05; FIG. 56). Theophyllineexhibited a similar synergy with HMB and leucine in adipocytes (FIGS. 57and 58), although no synergy was observed with resveratrol inadipocytes.

Theobromine is a naturally occurring methylxanthine found primarily incocoa and dark chocolate, as well as yerba mate and tea. Experimentswere conducted with a cocoa extract standardized to 12% theobromine;dose-response curves indicated concentrations below 0.1 μg/mL exert noeffect; accordingly, this was the concentration used in synergyexperiments. Combining cocoa extract/theobromine with 5 μM HMB resultedin a 260% increase in fat oxidation (p=0.021), and the cocoaextract/theobromine combination with 0.5 mM leucine resulted in a 673%increase (p=0.00035) (FIGS. 59 and 60). Combining the cocoaextract/theobromine with resveratrol exerted no significant effect onfat oxidation (FIGS. 59 and 60).

Isobutylmethylxanthine (3-isobutyl-1-methylxanthine; IBMX) is a methylxanthine similar to caffeine. It serves as both an adenosine antagonistand a non-specific PDE inhbitor. IBMX dose-response curves indicateconcentrations below 50 nM exert no effect; accordingly, this was theconcentration used in synergy experiments. IBMX exhibited weak butstatistically significant synergy with HMB, but not leucine, instimulating fat oxidation (73% increase, p=0.05) and glucose utilization(66%, p=0.05) in myotubes.

These data demonstrate significant synergistic effects of a severalnaturally occurring polyphenols on fat oxidation and glucose utilizationwhen these polyphenols are combined with either HMB or leucine. Theseeffects occur at levels which produce no independent effects and whichare readily achievable via diet or supplementation. These effects,mediated via Sirt1 and AMPK signaling, are significantly more robust forseveral of the polyphenols than those we previously observed for a lowdose of resveratrol combined with either HMB or leucine and more robustthan effects observed by us and others for high dose resveratrol.Chlorogenic acid (a hydroxycinnamic acid) and its hydrolysis product,quinic acid, as well as compounds structurally related to chlorogenicacid (cinnamic acid, ferulic acid) exerted especially robust effects.Highly significant effects were also observed with the resveratrolmetabolite piceatannol as well as with a non-polyphenolic compound fromseaweed (fucoxanthin, a xanthophyll that exhibits a highly resonantstructure commonly observed in polyphenols). These effects can also berecapitulated with naturally occurring non-specific PDE inhibitors.Thus, moderate levels of leucine and HMB can be utilized in synergisticcombinations with a number of polyphenols and related compounds tostimulate AMPK and sirtuin signaling and achieve benefits comparable toor exceeding those found with high-dose resveratrol.

These data also demonstrate that leucine and HMB exhibit significantsynergies with pharmaceuticals that converge on the same signalingpathways, thereby conferring efficacy to otherwise non-therapeutic dosesof these drugs. This can be an effective strategy for decreasing thelevels of these drugs required to achieve therapeutic efficacy, therebyattenuating side effects and adverse events otherwise associated withthem.

Example 6 Synergistic Effects of Metformin withResveratrol-Hydroxymethylbutryate Blend on Insulin Sensitivity inDiabetic Mice

Eight to ten week-old male diabetic db/db mice(C57BLKS/J-lepr^(db)/lepr^(db)) were randomized into six treatmentgroups (as described below) with 10 animals/group and kept on their dietfor 2 weeks:

-   -   Group 1 (labeled “control group”): standard diet (AIN 93G) only    -   Group 2 (labeled “high Metformin” (here 300 mg/kg BW)): standard        diet mixed with 1.5 g Metformin/kg diet (calculation: average        food consumption=8 g/day, average BW=40 g, 300 mg×0.04 kg=12 mg        Metformin/day/8 g food=1.5 mg Met/g diet)    -   Group 3 (labeled “low Metformin” (here 150 mg/kg BW): standard        diet mixed with 0.75 g Metformin/kg diet    -   Group 4 (labeled “very low Metformin” (here 50 mg/kg BW):        standard diet mixed with 0.25 g Metformin/kg diet    -   Group 5 (labeled “low Metformin plus Resv and CaHMB”): standard        diet mixed with 0.75 g Metformin plus 12.5 mg Resveratrol and 2        g CaHMB/kg diet    -   Group 6 (labeled “very low Metformin plus Resv and CaHMB”):        standard-diet mixed with 0.25 g Metformin plus 12.5 mg of        Resveratrol and 2 g CaHMB/kg diet.

Animals were housed in polypropylene cages at a room temperature of22±2° C. and regime of 12 h light/dark cycle. The animals had freeaccess to water and their experimental food throughout the experiment.At the of the treatment period (2 weeks) all animals were fastedovernight and humanely euthanized the next morning, and blood andtissues were collected for further experiments as described below.

Insulin Tolerance Test (ITT):

Insulin tolerance tests were performed at 2 pm on day 7. The mice wereinjected with insulin (0.75 U/kg) in ˜0.1 ml 0.9% NaClintraperitoneally. A drop of blood (5 microliter) was taken from the cuttail vein before the injection of insulin and after 15, 30, 45, and 60min for the determination of blood glucose. Change in blood glucose overthe linear portion of the response curve was then calculated.

Insulin:

Blood Insulin in serum was measured via Insulin ELISA kit from Millipore(Cat. # EZRMI-13K).

Glucose:

Blood glucose was measured via Glucose Assay Kit from Cayman (Cat. #EZRMI-13K).

Statistical Analysis:

All data is expressed as mean±STD. Data was analyzed by one-way ANOVA,and significantly different group means (p<0.05) were separated by theleast significant difference test using SPSS (SPSS Inc, Chicago, Ill.).

Results

The high dose (300 mg/kg bw) reduced plasma insulin by 27% (from 62 to45 uU/mL, p<0.02, FIG. 61) and in the HOMA_(IR) index by 35% (from 29 to18 units, p<0.025, FIG. 62), but exerted no significant effect on plasmaglucose in these highly insulin resistant animals. However, there wereno significant effects on body composition. A low dose of metformin(here 150 mg/kg) and a very low dose (50 mg/kg) exerted no significantindependent effects on any variable studied. In contrast, combiningeither the low or very low dose of metformin with HMB resulted insignificant decreases in plasma insulin from 62 uU/mL to 43 uU/mL(p<0.02, FIG. 61) comparable to that seen with high dose metformin, andthere was no significant difference between the low metformin-HMB blendversus the very low metformin-HMB blend. Consistent with thisobservation, the HOMA_(IR) index decreased from 29 units on the controldiet to 19 on the low metformin-HMB blend and to 16 on the very lowmetformin-HMB blend (p<0.025, FIG. 62), reflecting an improvement ininsulin sensitivity comparable to that found with high dose metformin.This is also reflected in the results of the insulin tolerance test;animals on the control, low-dose or very low-dose of metformin exhibitedminimal changes in blood glucose in response to the insulin challenge(FIG. 63). In contrast, those on the standard metformin dose and thoseon either the low or very low dose of metformin combined with HMBexhibited ˜60 mg/dL decreases in blood glucose over the 30 minute linearportion of the response curve (p<0.02; FIG. 63). Moreover, themetformin-HMB blends reduced visceral adiposity (FIG. 64). Animals onthe control diet had a mean visceral fat mass of 4.5 g, and this was notaffected by metformin at any dosage in the absence of HMB. A low dose ofmetformin+HMB and a very low dose of metformin+HMB reduced visceral fatby ˜20%, to 3.8 and 3.6 g, respectively, (p<0.03; FIG. 64). Thesetreatments also reduced liver mass, from 2.78 g (control) to 2.35 g and2.41 g, respectively (p<0.05 for both, FIG. 65).

Example 7 Synergistic Cell Signalling Effects of Metformin withResveratrol-Hydroxymethylbutryate Blend on Insulin Sensitivity inDiabetic Mice

Six groups of mice are treated as in Example 6, including collection ofblood and tissues for further experiments as described below.

SIRT1 Activity:

SIRT1 activity in cell lysates is measured by using the SIRT1Fluorimetric Drug Discovery Kit (BML-AK555, ENZO Life SciencesInternational, Inc. PA, USA). In this assay, SIRT1 activity is assessedby the degree of deacetylation of a standardized substrate containing anacetylated lysine side chain. The substrate utilized is a peptidecontaining amino acids 379-382 of human p53 (Arg-His-Lys-Lys[Ac]), atarget of SIRT1 activity; SIRT1 activity is directly proportional to thedegree of deacetylation of Lys-382. Samples are incubated with peptidesubstrate (25 μM), and NAD⁺ (500 μM) in a phosphate-buffered salinesolution at 37° C. on a horizontal shaker for 45 minutes. The reactionis stopped with the addition of 2 mM nicotinamide and a developingsolution that binds to the deacetylated lysine to form a fluorophore.Following 10 minutes incubation at 37° C., fluorescence is read in aplate-reading fluorometer at an excitation wavelength of 360 nm and anemission wavelength of 450 nm. Resveratrol (100 mM) serves as a SIRT1activator and suramin sodium (25 mM) as a SIRT1 inhibitor; wellsincluding each are utilized as positive and negative controls in eachset of reactions. A standard curve is constructed using deactylatedsubstrate (0-10 μM). Data is normalized to cellular proteinconcentration measured via BCA-assay.

Mitochondrial Extraction from Cell Lysates:

Mitochondria from tissue are isolated and lysed by using theMitochondrial Isolation Kit from BioChain (Cat# KC010100).

Sirt3 Activity:

SIRT3 activity is measured by using the SIRT3 Fluorimetric DrugDiscovery Kit (ENZO, BML-AK557) after mitochondrial extraction from celllysates. This assay is similar to SIRT1 activity, but uses a differentamino acid sequence of p53 (317-320: Gln-Pro-Lys(Ac)), as substrate.This substrate is most efficiently deacetylated by SIRT3.

Insulin Signaling:

Total and phosphorylated Akt, GSK-3β, IGF-1R, IR, IRS-1, p70S6K andPRAS40 in tissue lysates are measured via the Luminex Kits “Akt PathwayTotal 7-Plex Panel” (Cat# LHO0002) and “Akt Pathway Phospho 7-PlexPanel” (Cat# LHO0001) from Invitrogen Life Science.

AMPK Activity:

AMPK activity in cell lysates is measured via the Non-Radioisotopic AMPKKinase Assay Kit from CycLex (Cat# CY-1182).

RNA Extraction:

The Ambion ToTALLY RNA isolation kit (Ambion, Inc., Austin, Tex., USA)is used to extract total RNA from tissue according to the manufacturer'sinstructions. The concentration, purity and quality of the isolated RNAare assessed by measuring the 260/280 ratio (1.8-2.0) and 260/230 ratio(close to 2.0) via Spectrophotometer.

Gene Expression:

Expression of 18S, Sirt1, Sirt3, PGC1-α, cytochrome c oxidase subunitVIIc (COX 7), mitochondrial NADH dehydrogenase, nuclear respiratoryfactor 1 (NRF1), uncoupling protein (UCP2 (adipocyte)/UCP3 (myocyte),and GLUT4 are measured via quantitative real-time PCR using an ABI 7300Real-Time PCR system (Applied Bioasystems, Branchburg, N.J.) with aTaqMan core reagent kit. All primers and probe sets are obtained fromApplied Bioasystems TaqMan Assays-on-Demand and utilized accordingly tomanufacturer's instructions. Pooled RNA from each cell type isserial-diluted in the range of 0.0156-50 ng and is used to establish astandard curve; total RNA for each unknown sample is also diluted inthis range. RT-PCR reactions are performed according to the instructionsof the ABI Real-Time PCR system and TaqMan Real Time PCR Core Kit.Expression of each gene of interest is then normalized using thecorresponding 18S rRNA quantitation. Data for each gene is presented asa ratio to 18S rRNA.

Example 8 Syngergistic Effects of Leucine and its Metabolites withPolyphenols on Irisin

Compounds at doses having no independent effects on fatty acid oxidationwere tested for synergistic combinatorial effects on fatty acidoxidation and on irisin secretion. Compounds used included resveratrol(200 nM), cinnamic acid (1 μM), Chlorogenic acid (0.5 μM), quinic acid(500 nM), caffeine (10 nM), leucine (0.5 mM), and HMB (5 μM). Asdescribed in Example 5, C2C12 myotubes treated with the indicatedcombinations of compounds were used to produce conditioned media, withwhich 3T3-L1 adiposytes were then treated. Fatty acid oxidation wasmeasured as in Example 5. Irisin levels were measured by Western blotand ELISA, both in conditioned media and in plasma from mice treatedaccording to Example 4.

Western Blot:

The FNDC5 antibody (which binds irisin) was obtained from Abcam(Cambridge, Mass.). C2C12 myotubes were treated as described in Example5, with the indicated compound combinations, and the media and cellularfractions were prepared as described by Bostrom et al (Nature (2012)481:463-468). Protein was measured by BCA kit (Thermo Scientific). ForWestern blot, 6 μg protein (media) or 25 μg (cell lysate) was resolvedon 4-20% gradient polyacrylaminde gels (Criterion precast gel, Bio-RadLaboratroies, Hercules, Calif.), transferred to PVDF membranes,incubated in blocking buffer (3% BSA in TBS) and then incubated withprimary antibody (FNDC5), washed and incubated with secondaryhorseradish peroxidase-conjugated antibody. Visualization andchemiluminescent detection was conducted using BioRad ChemiDocinstrumentation and software (Bio-Rad Laboratories, Hercules, Calif.)and band intensity was assessed using Image Lab 4.0 (Bio-RadLaboratories, Hercules, Calif.), with correction for background andloading controls. Purified FNDC5 (Abcam, Cambridge, Mass.) was used as apositive control in these blots. FNDC5 was detected at 26-28 kDA andirisin was detected at 22-24 kDA.

ELISA: Irisin was also detected in C2C12 incubation media from treatedcells and in mouse plasma using a commercial enzyme immunoassay kitobtaind from Phoenix Pharmaceuticals, Inc. (Burlingame, Calif.). Thiskit uses an irisin (FNDC5 [16-127] fragment) andstreptavidin-horseradish peroxidase detection on a microplate reader at450 nm.

Results:

In Vitro:

Conditioned media from cells treated with combinations of resveratroland leucine, resveratrol and HMB, cinnamic acid and leucine, andcinnamic acid and HMB increased fatty acid oxidation in adipocytes,which was not observed for the compounds when used alone (FIG. 66). Thecombinations of resveratrol with either leucine or HMB, cinnamic acidwith HMB, and chlorogenic acid with either leucine or HMB resulted insignificant increases in FNDC5 protein expression in muscle cells (FIG.67), while the individual components exerted no effect. Combining quinicacid with leucine also results in a significant increase in FNDC5expression (FIG. 71), and in irisin secretion into the media (FIGS. 72and 73), while the individual component exert no effect. Measurement ofirisin secretion into the media was measured by Western blot (FIGS. 68and 72) and by ELISA (FIG. 69). Combining resveratrol with leucineresulted in a 234% increase in irisin secretion, from 6.76 to 22.55ng/ml (p=0.008) and the resveratrol-HMB combination elicited a 122.5%increase (p=0.00001, FIGS. 68 and 69). Similarly, the cinnamicacid-leucine combination stimulated a 40% increase (p=0.0005), while thecinnamic acid-HMB combination did not significantly stimulate irisinrelease into the media (FIG. 68). Although the chlorogenic acidcombinations with leucine or HMB increased muscle FNDC5 proteinexpression, this combination resulted in only modest, non-significantincreases in irisin secretion into the media. However, a three-waycombination of chlorogenic acid, caffeine, and either leucine or HMBresults in significant increases in both FNDC5 protein expression inskeletal muscle cells and of irisin secretion into the media (FIG. 74).

In Vivo:

Supplementing the diets of diet-induced obese mice with a low dose ofresveratrol (12.5 mg/kg diet) and a two-fold increase in dietary leucine(from 1.21 to 2.42%) for six weeks resulted in an 86% increase in plasmairisin, from 200±23 to 372±49 ng/ml (p=0.03, FIG. 70). The low-dose ofresveratrol exerted no independent effect on this variable.

These data indicate that, in addition to direct stimulation of sirtuinsignaling and fatty acid oxidation in skeletal muscle and fat cells,leucine-polyphenol combinations also stimulate irisin release fromskeletal muscle, thereby further promoting adipocyte fat oxidation.

Example 9 Signaling of BCAA Plus Sirtuin Pathway Activator CompositionsThrough Irisin In Vitro

C2C12 myotubes are treated a branched chain amino acid or a metabolitethereof (BCAA), a sirtuin pathway activator, both of these, or notreatment as a control, as in Example 5. The branched chain amino acidcan be leucine, such as at 0.5 mM. The sirtuin pathway activator can beresveratrol, such as at 200 nM. Myotubes are treated according to one ormore of the experiments described in Example 5 to produce conditionedmedia. Conditioned media is split into two samples, one that isuntreated and one that is treated to reduce or remove irisin. Treatmentto remove or inactivate irisin can include immunoprecipitation ofirisin, addition of an irisin-neutralizing antibody, or size exclusion(e.g. by filtration or a co-culture of myotubes with adipocytes, wherethe two cell types are separated by a membrane having a pore sizesmaller than the size of irisin). Measurement of the mRNA and/or proteinlevels of FNDC5 (irisin precursor protein) is used to confirm increasedFNDC5 expression, synergistically induced by treatment with thecombination composition. Measurement of irisin levels before and aftertreatment of the conditioned media (such as by Western blot or ELISA asdescribed in Example 8) is used to confirm increased irisin productionin untreated media and degree of reduction in the treated media. 3T3-L1adiposytes are then treated with either treated or untreated conditionedmedia. An output of mitochondrial biogenesis is then measured, such asfatty acid oxidation, glucose utilization, oxygen consumption,mitochondrial mass, or one or more indicia of fat cell browning (e.g.expression of browning-associated genes, such as Ucp1; and increase infatty acid oxidation). Gene expression can be measured as in Example 4.Other measures of mitochondrial biogenesis can be measured as in Example5. It is expected that combinations of BCAA (or BCAA metabolites) andsirtuin pathway activators synergistically increase irisin expression,and that adipocytes exposed to untreated media (as in Example 5) willshow signs of synergistic increases in mitochondrial biogenesis. It isfurther expected that treatment of the conditioned media to remove orinactivate irisin from conditioned media giving a synergistic effectwill significantly reduce the mitochondrial biogenesis otherwiseobserved. Such a result will indicate that combination compositions ofthis kind produce at least a portion of their synergistic effectsthrough irisin signaling.

Example 10 Signaling of BCAA Plus Sirtuin Pathway Activator CompositionsThrough Irisin In Vivo

Six week old male c57/BL6 mice were fed a high-fat diet with fatincreased to 45% of energy (Research Diets D12451) for 6 weeks to induceobesity. At the end of this obesity induction period, animals wererandomly divided into seven different diet treatment groups with 10animals per group (overall 70 animals) and maintained on these diet for6 weeks, according to groups in Example 4. Measurements, samples, andtissues were collected as in Example 4. Tissues collected includedsubcutaneous adipose tissue, which is ordinarily a white adipose tissue.RNA was extracted from the subcutaneous adipose tissue, and geneexpression was evaluated according to methods described in Example 4.Gene expression analysis included determining the expression levels ofPGC1α (a known activator of FNDC5 expression, which is the precursor ofirisin) and UCP1 (a brown-fat-selective gene, an increase in expressionof which is stimulated by irisin). Plasma levels of irisin were alsoevaluated, the results of which are described in Example 8 andillustrated in FIG. 70. Mice may also evaluated for increases in otherindicators of induction of fat cell browning, including increasedexpression in subcutaneous fat and other adipose tissue of one or moreother brown-fat-selective genes (e.g. Cidea, Prdm16, and Ndufs1).Protein expression may also be determined by Western blot or ELISA, asin Example 8. Fat cell browning is also indicated by an increase infatty acid oxidation in fat cells.

Minimal UCP1 expression was detected in white subcutaneous adiposetissue in control mice. However, supplementing the diets of thesediet-induced obese mice with a low dose of resveratrol (12.5 mg/kg diet)and a two-fold increase in dietary leucine (from 1.21 to 2.42%) for sixweeks resulted in a 344% increase in UCP1 expression (p<0.05, FIG. 75).The low dose of resveratrol exerted no independent effect on thisvariable, and the HMB-resveratrol combination exerted no detectableeffect. Consistent with these observations, there was a correspondingtwo-fold upregulation of PGC1α expression in white adipose tissue inresponse to feeding the resveratrol-leucine combination (p=0.04, FIG.76).

These data indicate that, in addition to direct stimulation of sirtuinsignaling and fatty acid oxidation in skeletal muscle and fat cells, theresveratrol-leucine combination also stimulates browning of whiteadipose tissue. This correlates with our observation that theleucine-resveratrol combination stimulates irsin release from muscleboth in vitro and in vivo (see, e.g., Example 8).

It should be understood from the foregoing that, while particularimplementations have been illustrated and described, variousmodifications can be made thereto and are contemplated herein. It isalso not intended that the invention be limited by the specific examplesprovided within the specification. While the invention has beendescribed with reference to the aforementioned specification, thedescriptions and illustrations of the preferable embodiments herein arenot meant to be construed in a limiting sense. Furthermore, it shall beunderstood that all aspects of the invention are not limited to thespecific depictions, configurations or relative proportions set forthherein which depend upon a variety of conditions and variables. Variousmodifications in form and detail of the embodiments of the inventionwill be apparent to a person skilled in the art. It is thereforecontemplated that the invention shall also cover any such modifications,variations and equivalents.

What is claimed is: 1.-30. (canceled)
 31. A composition comprising: a)at least about 500 mg of leucine and/or at least about 200 mg of one ormore metabolites thereof, wherein the one or more leucine metabolitesare selected from the group consisting of keto-isocaproic acid (KIC),alpha-hydroxy-isocaproic acid, and HMB; and b) an anti-diabetic agentcomprising a thiazolidinedione.
 32. The composition of claim 31, whereinthe composition is substantially free of the individual amino acidsalanine, glutamic acid, glycine, isoleucine, valine, and proline. 33.The composition of claim 31, wherein when (a) is leucine, the molarratio of (a) to (b) is greater than
 1000. 34. The composition of claim31, wherein when (a) is HMB, the molar ratio of (a) to (b) is greaterthan
 10. 35. The composition of claim 31, wherein the compositioncomprises a subtherapeutic amount of the anti-diabetic agent.
 36. Thecomposition of claim 31, wherein the anti-diabetic agent comprisesrosiglitazone.
 37. The composition of claim 31, wherein whenadministered to a subject, the anti-diabetic agent comprisesrosiglitazone in an amount effective to achieve a circulating level ofat least about 1 nM rosiglitazone in the subject.
 38. The composition ofclaim 31, wherein the composition is substantially free of an individualnon-branched chain amino acid.
 39. The composition of claim 31, furthercomprises a sirtuin pathway activator that activates one or more ofSIRT1, SIRT3, AMPK, and PGC1α.
 40. The composition of claim 31, furthercomprising at least one of a hydroxycinnamic acid, a stilbene, apolyphenol or a polyphenol precursor.
 41. The composition of claim 40,wherein the polyphenol or polyphenol precursor is selected from thegroup consisting of chlorogenic acid, resveratrol, caffeic acid,cinnamic acid, ferulic acid, piceatannol, ellagic acid, epigallocatechingallate, grape seed extract, and any analog thereof.
 42. The compositionof claim 31, further comprises a phosphodiesterase inhibitor.
 43. Thecomposition of claim 31, further comprises a resveratrol.
 44. Thecomposition of claim 31, wherein the composition is formulated as a unitdose.
 45. The composition of claim 31, wherein the composition isformulated as a tablet, capsule, or gel capsule.
 46. A method oftreating diabetes in a subject in need thereof comprising: administeringto the subject the composition of claim 31, wherein the insulinsensitivity in the subject is increased.
 47. The method of claim 46,wherein the anti-diabetic agent comprises rosiglitazone.
 48. The methodof claim 47, wherein the rosiglitazone is an amount effective to achievea circulating level of at least about 1 nM rosiglitazone in a subject.49. The method of claim 46, wherein fat oxidation in the subject isincreased or inflammatory response in the subject is decreased.
 50. Thecomposition of claim 46, wherein the composition is substantially freeof the individual amino acids alanine, glutamic acid, glycine,isoleucine, valine, and proline.
 51. The method of claim 46, wherein thecomposition is substantially free of an individual non-branched chainamino acid.
 52. The method of claim 46, wherein the compositioncomprises a subtherapeutic amount of the anti-diabetic agent.
 53. Themethod of claim 46, wherein the composition further comprises a sirtuinpathway activator that activates one or more of SIRT1, SIRT3, AMPK, andPGC1α.
 54. The method of claim 46, wherein the composition furthercomprises a resveratrol.
 55. The method of claim 46, wherein each of (a)and (b) are formulated as a tablet, capsule, or gel capsule.